Dna markers for management of cancer

ABSTRACT

A method is provided for assessing allelic losses and hypermethylation of genes in CpG tumor promotor region on specific chromosomal regions in cancer patients, including melanoma, neuroblastoma breast, colorectal, and prostate cancer patients. The method relies on the evidence that free DNA and hypermethylation of genes in CpG tumor promotor region may be identified in the bone marrow, serum, plasma, and tumor tissue samples of cancer patients. Methods of melanoma, neuroblastoma, colorectal cancer, breast cancer and prostate cancer detection, staging, and prognosis are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/725,309, filed Mar. 16, 2010, which is a continuation of U.S. patentapplication Ser. No. 10/809,965, filed Mar. 25, 2004, which claimspriority to U.S. Provisional Application Ser. No. 60/457,395, filed Mar.25, 2003. The content of the three above-mentioned applications isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with support in part by grants from NCI (GrantNos. R21CA100314, PO CA 29605 Project II, and PO CA 13917 Project II),Gonda Foundation, USA DOD Breast Cancer Research Grant, CaliforniaBreast Cancer Research Grant, and Roy E Coates Foundation. Therefore,the U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the fields of molecular biology andoncology and provides methods for diagnosis, staging and monitoring ofcancer patients.

BACKGROUND OF THE INVENTION

Bone Marrow is the most frequent site of the systemic spread of sometypes of cancer, including breast (Abrams et al 1950 and Lee 1983),neuroblastoma, colorectal, and prostate cancer. Once metastases areclinically apparent, overall prognosis is poor. Undetected occult tumorcells contribute to disease recurrence and therefore methods to identifysubclinical disease (micrometastasis) may improve staging and guideadditional therapeutic decisions. Historically, conventional cytologicassessment of blood and bone marrow (BM) aspirates has been performedwith limited success (Molino et al 1991 and Beiske et al 1992).Immunocytochemical techniques using antibodies specific to epithelialantigens have improved sensitivity and can identifying a single tumorcell amongst a background of >1 million normal cells (Osborne et al 1989and Chaiwun et al 1992). Enrichment methods with antibody-magnetic beadconjugates of BM aspirates have demonstrated the presence of occulttumor cells in early stage breast cancer patients (Osborne et al 1989and Rye et al 1997).

Recently it has been shown that the detection of micrometastasis in theBM of early stage breast cancer patients is an independent prognosticrisk factor (Braun et al 1998 and Diel et al 1996). However, theaccurate microscopic analysis of many cytologic samples requiresconsiderable cytopathologic expertise and can be tedious, particularlyif performed serially to assess disease progression and/or response totreatment. Additionally, the variable specificity of individualantibodies used to detect single cells has been questioned (Braun et al1998; Litle et al 1996; and Moll et al 1982). Finally, these assaysystems cannot characterize the biologic behavior of the cells beingdetected and thus many may represent dormant tumor cells, apoptoticcells, nonpathologic tumor cells, or displaced normal breast epithelialcells.

A variety of serial genetic changes have been implicated in theinitiation and progression of solid tumors. One such event, allelicimbalance (loss of heterozygosity; LOH) has been shown to occur commonlyin primary breast tumors and with additional frequency in metastasis(Takita et al 1992; Hampl et al 1999; Driouch et al 1997; and Silva etal 1999). Furthermore, there is emerging evidence to suggest thatmicrosatellite markers for detecting LOH at specific chromosome loci mayhave important clinical prognostic correlations (Takita et al 1992;Hampl et al 1999; Emi et al 1999; and Hirano et al 2001). However, theexamination of an excised primary tumor specimen may be of limited valuein that it provides information of those genetic events that haveoccurred and not ongoing alterations which may be of clinical relevance,either prognostically or for therapeutic decisions. Additionally,because of the potentially long latent period that may exist betweenearly breast cancer diagnosis and clinically detectable systemicrecurrence, improved assessment methods are needed for serialsurveillance of occult disease progression and monitoring response totherapy.

Recently it has been shown that free tumor-associated DNA can beidentified in the serum and plasma from patients with melanoma, breast,lung, renal, gastrointestinal, and head and neck tumors (Anker et al1997; Chen et al 1999; Chen et al 1996; Silva et al, Cancer Research1999; Shaw et al, 2000; Taback et al, Academy of Science 2001; Taback etal, Cancer Research 2001; Sanchez-Cespedes et al 1998; Goessl et al,Cancer Research 58 1998; Fujiwara et al 1999; Hibi et al 1998; Kopreskiet al 1997; Mayall et al 1999; Nawroz et al 1996; and Stroun et al1987). Furthermore a high-quality concordance has been shown to existbetween the genetic alterations (i.e., LOH, microsatellite instability,mutations) found in circulating tumor DNA and those from the primarytumor suggesting a potential surrogate tumor marker (Chen et al 1996;Silva, Cancer Research 1999; Shaw et al 2000; Fujiwara et al 1999; andHibi et al 1998). Early studies have shown the prognostic importance ofcirculating microsatellite markers for LOH in blood (Silva, CancerResearch 1999 and Taback et al, Cancer Research 2001). Although, BM is acommon site for recurrence of some types of cancer, such as breast andprostate cancer, to date, BM has not been studied for the presence ofsuitable genetic markers.

Recently, methylation of gene promoter regions and the role that thisepigenetic event plays in the development of various cancers has becomean important area of investigation in assessing the mechanisms of tumorsuppressor and regulatory gene inactivation. The tumor suppresor genes(TSG) can be transcriptionally silenced when their promoter region CpGislands contain methylated cytosines located 5′ to an adjacent guanine.The utilization of methylation-specific PCR (MSP) assay has simplifiedand significantly improved detecting hypermethylated CpG bases withminimum amount of DNA. The methylation status of several TSG promoterregions have been profiled for a number of cancers. The hypermethylationof CpG islands of promoter regions of TSG is quite common and is asignificant genetic aberration for tumor cells to shut off TSGexpression.

Majority of these studies have been focused on carcinomas; there arelimited studies in cutaneous melanomas and other types of cancers. Thestudies of epigenetic inactivation of TSG in melanomas have been limitedmostly to methylation of promoter regions of p16^(INK4a) andMGMT(O⁶-methylguanine-DNA methyltransferase). Interestingly, thefrequency of TSG inactivation or mutations in oncogenes is reportedlylow in cutaneous melanomas. These observations have been a major enigmain deciphering the genetic events occurring in melanoma progression.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery that DNAmarkers can be detected in cell-free bone marrow samples and are usefulfor cancer diagnosis, staging, and prognosis.

Accordingly, the invention features a method of detecting DNA markers ina sample, comprising providing a cell-free bone marrow sample from asubject and detecting one or more DNA markers in the sample. Examples ofDNA markers include those in the 1p, 3p, 6p, 6q, 8p, 10q, 11q, 14q, 16q,or 17p region. In particular, the DNA markers may be indicative of LOH,DNA hypermethylation, or DNA mutation. Such DNA markers include D1S228,D8S321, D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421, D16S422,D17S796, D17S849, D17S855, D18S58, D18S61, and D18S70; those indicativeof hypermethylation in RASSF1A, MGMT, GSTP1, RAR-β, TWIST, APC, DAPK,P16, or Cyclin D2 promoter; and those indicative of mutation in KRAS andBRAF gene (e.g. mutation at codon 12 of KRAS and BRAF K600E mutation).

In one aspect, the invention provides a method of detecting cancer,comprising providing a cell-free bone marrow sample from a subject anddetecting one or more DNA markers in the sample, wherein LOH,hypermethylation, or mutation of the markers is indicative of cancer(e.g., melanoma, neuroblastoma, colorectal, breast, or prostate cancer)in the subject.

In another aspect, the invention provides a method of staging cancer,comprising providing a cell-free bone marrow sample from a subjectsuffering from cancer and detecting one or more DNA markers in thesample, wherein LOH, hypermethylation, or motation of the markers isindicative of an advanced stage of the cancer in the subject.

The invention further provides a method of prognosing cancer, comprisingproviding a cell-free bone marrow sample from a subject suffering fromcancer and detecting one or more DNA markers in the sample, wherein LOH,hypermethylation, or mutation of the markers is indicative of a poorprognosis of the cancer in the subject.

The invention is also based on the unexpected discovery that LOH andhypermethylation of DNA markers, when combined, provide more sensitiveand precise diagnosis, staging and prognosis of cancer than when usedindividually. Therefore, the invention provides a method of detectingLOH and DNA hypermethylation, comprising providing a sample from asubject and detecting a combination of LOH and DNA hypermethylation inthe sample (e.g., a serum, plasma or tissue sample). In one embodiment,the LOH is detected through DNA markers including D1S228, D8S321,D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421, D16S422,D17S796, D17S849, D17S855, D18S58, D18S61, or D18S70. In anotherembodiment, the DNA hypermethylation is detected in RASSF1A, MGMT,GSTP1, RAR-β, TWIST, APC, DAPK, P16, KRAS, BRAF, or Cyclin D2 promoter.

In one aspect, the invention features method of detecting cancer,comprising providing a sample from a subject and detecting one or moreDNA markers in the sample, wherein a combination of LOH andhypermethylation of the markers is indicative of cancer (e.g., melanoma,neuroblastoma, colorectal, breast, or prostate cancer) in the subject.

In another aspect, the invention features a method of staging cancer,comprising providing a sample from a subject suffering from cancer anddetecting one or more DNA markers in the sample, wherein a combinationof LOH and hypermethylation of the markers is indicative of an advancedstage of the cancer in the subject.

In still another aspect, the invention features a method of prognosingcancer, comprising providing a sample from a subject suffering fromcancer and detecting one or more DNA markers in the sample, wherein acombination of LOH and hypermethylation of the markers is indicative ofa poor prognosis of the cancer in the subject.

Moreover, the invention provides kits and packaged products forimplementing the methods described above. For example, one packagedproduct comprises a container, one or more agents for detecting one ormore DNA markers in a sample and an insert associated with the containerand indicating that the sample is a cell-free bone marrow sample.

Another example of a packaged product comprises a container, one or moreagents for detecting one or more DNA markers in a cell-free bone marrowsample from a subject, and an insert associated with the container andindicating that LOH, hypermethylation, or mutation of the markers isindicative of cancer in the subject.

A packaged product may also comprise a container, one or more agents fordetecting one or more DNA markers in a cell-free bone marrow sample froma subject suffering from cancer, and an insert associated with thecontainer and indicating that LOH, hypermethylation, or mutation of themarkers is indicative of an advanced stage of the cancer or a poorprognosis of the cancer in the subject.

The invention further provides (1) a kit comprising one or more agentsfor detecting a combination of LOH and DNA hypermethylation of one ormore DNA markers in a sample; (2) a packaged product, comprising acontainer, one or more agents for detecting one or more DNA markers in asample from a subject and an insert associated with the container andindicating that a combination of LOH and hypermethylation of the markersis indicative of cancer in the subject; and (3) a packaged product,comprising a container, one or more agents for detecting one or more DNAmarkers in a sample from a subject suffering from cancer, and an insertassociated with the container and indicating that a combination of LOHand hypermethylation of the markers is indicative of an advanced stageof the cancer or a poor prognosis of the cancer in the subject.

The methods of the present invention advantageously permit a minimallyinvasive detection of tumor genetic changes that may provide valuableprognostic and diagnostic information, which may improve staging of thedisease and monitoring of disease progression and response to therapy.In addition, because the methods of the present invention may be used tosurvey ongoing genetic changes, they may also be used to identifypotential targets to individualize patient therapy. The method may alsobe used to identify markers in BM aspirates, plasma, and serum for othertypes of cancers.

The above-mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows representative examples of paired frozen andparaffin-embedded melanoma tumors analyzed by CAE for determiningmethylation status of (A) RAR-β2, (B) MGMT, and (C) RASSF1A. Methylated(M) and unmethylated (U) PCR products from frozen (F1) orparaffin-embedded (P1) tumor specimens were analyzed simultaneously anddistinguished by size and fluorescence.

Also shown are representative examples of tumor histopathology negativepatients' paraffin-embedded lymph nodes (PLN) analyzed by CAE fordetermining methylation status of (D) RAR-β2, (E) MGMT, and (F) RASSF1A.

FIG. 2 shows representative expression and re-expression of RAR-β2,RASSF1A and MGMT in two melanoma cell lines treated with 5Aza-dC. Thecells were treated for four days with different concentrations of5Aza-dC followed by 24 h treatment with ATRA where indicated. Geneexpression was analyzed by RT-PCR. The house-keeping gene GAPDH wasincluded as an RT-PCR control for all assays.

FIG. 3 shows representative PCR analysis of promoter region CpG islandsequence of MGMT and RAR-β2 from bisulfite-treated DNA obtained frommelanoma cell lines. Fully methylated CpGs are indicated as solid blackboxes and partially methylated CpGs are shown as shaded boxes. All CpGscontained in the MSP products are shown.

FIG. 4 provides representative images demonstrating LOH in breast cancerpatients' paired BM aspirate (BM) and primary tumors (T) at D14S62,D14S51, and D8S321, respectively. Allelic loss is represented by thearrows. The first lane of each panel exhibits patients' lymphocyte DNA(L) allele pattern as a control.

DETAILED DESCRIPTION OF THE INVENTION

Since BM is a common site for cancer recurrence in certain types ofcancer and because an application of conventional histochemicaltechniques to BM has been limited due to sub-optimal efficiency andsensitivity, it is one object of the present invention to determinewhether BM aspirates may be used as a source of tumor-specific DNAassociated with systemic metastasis from cancer, including metastasisassociated with neuroblastoma, breast, prostate, and colorectal cancer.It is a further object of the present invention to identifytumor-specific nucleic acid alterations in the bone marrow,serum/plasma, and tumor tissue samples of cancer patients as diagnosticand prognostic markers of malignancy. Also, since the hypermethylationof CpG islands of promoter regions of TSG appears to play a significantrole in the development of various cancers, it is another objective ofthe present invention to identify TSG and tumor-related genesmethylation of which could indicate development of a cancer. It is afurther object of the present invention to develop a method of using theidentified methylation markers in the bone marrow, serum/plasma, andtumor tissue samples of cancer patients to diagnose malignancy.

It is a discovery of the present invention that LOH may be detected inBM aspirates and that the advancement of AJCC stages is associated withan increased incidence of LOH. In one study, the inventors used a panelof microsatellite markers for LOH on chromosomes 1p, 3p, 6p, 6q, 8p,10q, 11q, 14q, 16q, and 17p to demonstrate the association between theLOH identified in BM aspirates with stage and tumor type in breastcancer. The inventors believe that other cancers that metastasizepreferentially in bone, such as melanoma, prostate, and colorectalcancers, may be detected and monitored using the same group of LOHmarkers that were identified in breast cancer patients.

In another study, the inventors have demonstrated a correlation of LOHidentified in serum/plasma of prostate cancer patients and AJCC staging.In still another study, the inventors showed that the presence ofcertain circulating nucleic acids in serum/plasma may assist indiagnosis of colorectal cancer. Accordingly, the present inventionprovides tumor-related genetic markers in BM aspirates, serum/plasma,and tumor tissue samples of cancer patients and provides a uniqueapproach for assessing the subclinical systemic disease progression andthe monitoring of cancer patients. The present invention also providesmolecular techniques for the identification of genetic alterations oncirculating nucleic acids in the bone marrow aspirates, plasma, serum,and tumor tissue of cancer patients.

One aspect of the present invention provides a detection assay fordetecting the loss of heterozygosity (LOH) in DNA from BM, tumor tissue,plasma, and serum. The assay comprises the steps of (a) amplifyingnucleic acid from an LOH marker, if present, (b) detecting the presenceor absence of the LOH marker, and (c) correlating the findings with theoccurrence and/or progression of a cancer. The determination ofheterozygosity is well within the skill of the art and includesexamining the second sample of DNA, which is isolated fromnon-neoplastic tissue. For example, U.S. Pat. No. 6,465,177, which isassigned to the assignee of the present invention and the content ofwhich is incorporated herein by the reference, describes the detectionof the loss of heterozygosity in the tumor and serum of melanomapatients.

Although any detection method may be used in association with markers ofthe present invention, in one embodiment amplification/detection methodsused were PCR-based methods selected from the group consisting of PCRand gel electrophoresis using labeled primers (fluorescent orradioactive), RealTime PCR using specific labeled primers Taqman andprobes (labeled with chromatographic dyes), and capillary arrayelectrophoresis (CAE) with labeled PCR primers (no probes).

In one embodiment, the detection is carried out in a sample derived frombone marrow aspirates, plasma/serum, or tumor tissues. In anotherembodiment, because the combination of assessing blood, tumor tissue,and bone marrow is believed by the inventors to have a better predictiveand diagnostic value, LOH is assessed in several different samplesselected from the group consisting of BM aspirates, tumor tissue, serum,and plasma.

In one embodiment of the present invention, the set of alleles which aretested for LOH in BM aspirate, blood/plasma, or tumor tissue sample areselected from the group consisting of D1S228, D8S321, D4S175, D4S1586,D5S299, D8S133, D8S261, D8S262, D8S264, D9S171, D10S197, D10S591,D10S532, D14S51, D14S62, D15S127, D16S421, D16S422, D17S796, D17S849,D17S855, D18S58, D18S61, and D18S70.

It is also a discovery of the present invention that the methylation ofDNA CpG tumor promoter regions is detectable in the plasma/serum, tumortissue, and bone marrow of breast melanoma colon cancer patients for thefollowing genes: RASSF1A, MGMT, GSTP1, RAR-β, TWIST, APC, DAPK, P16, andCyclin D2 (CCND2). For example, although there are no reportedcomprehensive studies on melanoma tumor methylation correlating withclinicopathology, the inventors discovered an inactivation of a newlyidentified TSGs RASSF1A and RAR-β in melanomas, as well as methylationof MGMT.

The inventors believe that methylation markers may be used to providesignificant prognostic and diagnostic information in cancer patients,including melanoma, colorectal, breast, and prostate cancer patients.The inventors also believe that utilization of both LOH markers and DNAmethylation markers will allow establishing a comprehensive panel ofhuman genetic prognostic molecular markers (PMMs) for melanoma,colorectal, breast, and prostate cancer. Primary tumors, metastatictissue, blood (plasma/serum) or/and BM may be tested for methylation andmicrosatellite DNA markers for diagnosis and prognosis. For example,blood (plasma/serum) and BM LOH markers may be used as PMM in patientfollow up to identify sub-clinical disease recurrence. Assessment oftumor tissue may be used for prognosis of disease outcome. Although itappears that LOH and methylation markers somewhat overlap for breast,prostate, melanoma and colon cancer, there are specific LOH andmethylation markers that are more frequent or exclusively in specificcancers. Thus, in one embodiment, a panel of markers specific to thecancer suspected in the patient is used. In another embodiment, a panelcomprising a broad range markers, including non-specific markers, isused to conduct a broader screening for various types of cancer.

In another aspect of the present invention, BM aspirates, blood, andtumor are assessed collectively for LOH and methylation markers toobtain a comprehensive profile of cancer patients, including predictionof metastasis to lymph nodes and disease outcome. The obtained resultsmay be used to predict metastasis to lymph nodes (sentinel node) anddisease outcome in cancer patients, including breast cancer, prostatecancer, and melanoma patients.

For example, in one embodiment, BM aspirates, serum, and/or plasmasamples are evaluated for the presence of microsatellite markersselected from the group consisting of D1S228, D8S321, D4S175, D4S1586,D5S299, D8S133, D8S261, D8S262, D8S264, D9S171, D10S197, D10S591,D10S532, D14S51, D14S62, D15S127, D16S421, D16S422, D17S796, D17S849,D17S855, D18S58, D18S61, and D18S70 and methylation markers selectedfrom the group consisting of RASSF1A, MGMT, GSTP1, RAR-β, TWIST, APC,DAPK, P16, and Cyclin D2 (CCND2) to obtain a prognostic and diagnosticinformation in cancer patients, including melanoma, breast, colorectal,and prostate cancer patients. In another embodiment, BM aspirates,serum, and/or plasma samples are evaluated for the presence ofmicrosatellite markers with LOH on chromosomes 1p, 3p, 6p, 6q, 9p, 10q,11q, and 12q and methylation markers RASSF1A, MGMT, and RAR-β to obtainprognostic and diagnostic information in breast cancer patients.

There is mounting evidence to suggest that the presence of occult tumorcells in the BM of breast cancer patients may have prognosticsignificance (Diel et a 1996; Mansi et al 1991; Berger et al 1988; Coteet al 1991; Dearnaley et al 1991; Harbeck et al 1994; and Braun et al, NEngl J Med 2000). Furthermore, some have shown these findings to beindependent of pathologic lymph node status (Braun et al, J Natl CancerInst 1998 and Diel et al 1996). These studies are important because,historically, 20% of lymph node negative patients will subsequentlydevelop systemic disease and therefore, early detection of BMmicrometastasis may identify high-risk patients for additional systemictherapy. More so, BM provides a readily accessible source to seriallymonitor subclinical disease progression and the potential impact ofadjuvant therapies early in the disease course. Conventional histologicanalysis of BM aspirates for tumor cells has proven unreliable (Molinoet al 1991 and Beiski et al 1992). More recently, immunocytochemicaltechniques using antibodies to epithelial antigens expressed on tumorcells have improved detection sensitivity. However, assay reliabilityhas been shown to be highly dependent on the antibody selected as wellas the variability by which the tumor cell expresses the preferredepitope (Braun et al, J Natl Cancer Inst 1998 and Moll et al 1982.Finally, sample processing and antibody staining require considerableattention to methodology and an experienced reviewer to interpret theresults.

With the implication of an accrual of aberrant genetic events in tumordevelopment and progression, and their potential for clonality, thesegenetic markers may provide unique surrogates for monitoring subclinicaldisease events, particularly in light of the ease and widespread use ofPCR techniques. Studies have demonstrated the presence of circulatingnucleic acids in the plasma and serum of patients with variousmalignancies (Stroun et al 1987). In breast cancer, LOH presence inplasma/serum has been described to occur anywhere from 15% to 66% (Chenet al, Clin Cancer Res 1999; Silva et al, Cancer Res 1999; Shaw et al2000; Taback et al, Ann NY Acad Sci 2001; and Mayall et al 1999). Theseresults may vary due to differences in the techniques of samplecollection and processing, DNA isolation, PCR methods, and scoring ofLOH. Furthermore, in the earlier work, the inventors have shown that thepresence of the circulating tumor DNA increases with the advancing stageof disease (Taback et al, Ann NY Acad Sci 2001). Since BM is a frequentsite of melanoma, prostate, colorectal and breast cancer relapse, it wasan object of the present invention to determine whether BM aspiratesharbor tumor-specific DNA alterations associated with early breastcancer progression.

The present invention provides highly sensitive methods of detectingtumor-specific DNA in the BM aspirates, plasma/serum, and tumor tissueof cancer patients, including melanoma, breast, colorectal, and prostatecancer patients. The increased incidence in the more advanced stagescorrelates with tumor burden and therefore may have applicability as asurrogate marker for disease detection, prognosis, and monitoring tumorprogression and response to therapy. The present invention demonstratesan association between known prognostic factors in breast cancer (tumorhistopathology, tumor size, lymph node status, and AJCC stage) and anincidence of LOH and methylation markers in BM, blood (plasma/serum),and primary tumor and metastatic tissues.

Some advantages of the methods of the present invention overconventional methods include the ease of their use, high sensitivity andspecificity, and their broad application to a variety of malignancies.Additional tumor-specific genetic markers or combinations thereof may beeasily incorporated into methods of the present invention to furtherenhance the assay's utility.

The methods of the present invention may provide a uniquealternative/supplement to optical systems for occult tumor detectionwhich can be technically demanding and viewer dependent. The methods ofthe present invention may also provide an alternative/supplement toRT-PCR methods that assess mRNA markers which may have limitedspecificity as a result of unstable gene products, variable expressionlevels, and nonspecific transcripts (Zippelius et al 1997; Bostick et al1998; Ko et al 1998; and Jung et al 1998). The inventors believe thatthe detection of genomic alterations in BM may offer more specificitythan immunohistochemical and/or current mRNA marker assays.

In one study, which is described in more detail in Example 5, theinventors observed LOH in BM ranging from 0% to 12% for variousmicrosatellite markers (see Table 10). A similar detection of LOH hasbeen described from the peripheral plasma/serum of early stage breastcancer patients (Chen et al, Clinical Cancer Research 1999; Shaw et al2000; and Taback, Ann NY Acad of Sci 2001). For 10 of the 11 patientswhose BM contained LOH, primary tumor blocks were available forassessment and in all cases, a similar corresponding LOH pattern wasidentified in the respective primary tumor specimens. The findingsdemonstrate the specificity of this marker detection system.

In the study discussed in Example 5, no patients had detectable tumorscells identified on routine histopathologic examination. Thisdemonstrates the relative ease and sensitivity that the methods of thepresent invention provide in the identification and diagnosis ofsubclinical disease. Because of the earlier detection of breast cancersand the benefits of adjuvant radiotherapy, immunotherapy, andchemotherapy in these stages, the methods and microsatellite markers ofthe present invention provide improved occult disease surveillance andability to assess individual patient risk more accurately. This allowsmodification of treatment strategies before clinical manifestationsoccur.

Breast cancer recurrence is a result of undetected metastasis at thetime of primary patient treatment. More sensitive methods are needed toidentify subclinical disease progression to better accompany thoseincreasing advances in early breast cancer screening. Aberranthypermethylation of tumor-suppressor genes is found frequently inprimary breast tumors and has been implicated in disease initiation andprogression. The increased sensitivity for the detection of methylatedgenes associated with a cancer phenotype among a background ofunmethylated genes from normal cells offers a potential specificsurrogate marker for molecular detection of occult disease progression.We evaluated whether tumor-associated methylated DNA markers could beidentified circulating in BM aspirates and paired serum samples from 33early-stage patients undergoing surgery for breast cancer. Methylationspecific PCR was performed using a tumor-related gene panel for RAR-β2,MGMT, RASSF1A and APC. Tumor-associated hypermethylated DNA wasidentified in 7 (21%) of 33 BM aspirates and 9 (27%) serum samples. Inthree patients, the bone marrow and serum were positive forhypermethylation. The most frequently detected hypermethylation markerwas RASSF1A occurring in 7 (21%) patients. Concordance was presentbetween gene hypermethylation detected in BM/serum samples andmatched-pair primary tumors. Advanced AJCC stage was associated with anincreased incidence of circulating gene hypermethylation. This studydemonstrates the novel finding of tumor-associated epigenetic markers inBM aspirates and their potential role as targets for molecular detectionand as an aid to early-stage breast cancer patient risk identification.

Gene promoter region hypermethylation is a frequent event in primarybreast cancer. However its impact on tumor progression and potentialprognostic implications remain relatively unknown. We conductedhypermethylation profiling of 151 primary breast tumors with associationto known prognostic factors in breast cancer using methylation specificPCR for six known tumor suppressor and related genes: RASSF1A, APC,Twist, CDH1, GSTP1 and RAR-β2. Furthermore correlation with sentinellymph node tumor status was assessed as it represents the earliest stageof metastasis that can be readily detected. Hypermethylation for any onegene was identified in 147 (97%) of 151 primary breast tumors. The mostfrequently hypermethylated gene was RASSF1A (81%). Hypermethylation ofthe CDH1 was significantly associated with primary breast tumorsdemonstrating lymphovascular invasion (p=0.008), infiltrating ductalhistology (p=0.03), and negative for the estrogen receptor (p=0.005),whereas RASSF1A and RAR-β2 gene hypermethylation were significantly morecommon in ER positive (p<0.001) and HER2 positive (p<0.001) tumors,respectively. In multivariate analysis, hypermethylation of GSTP1 and/orRAR-β2 was significantly associated with patients have macroscopicsentinel lymph node metastasis, odds ratio 4.59 (95% CI, 2.02 to 10.4;p<0.001). Hypermethylation profiling of primary breast cancers may haveclinical and pathologic utility for assessing patient prognosis andpredicting early lymph node regional metastasis.

Aberrant methylation of CpG islands in promoter regions of tumorsuppressor genes (TSG) has been demonstrated in epithelial origintumors. However, the methylation profiling of tumor-related genepromoter regions in cutaneous melanoma tumors has not been reported.Seven known or candidate TSGs that are frequently hypermethylated incarcinomas were assessed by methylation-specific polymerase chainreaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanomatumors. Four TSGs were frequently hypermethylated in 86 metastatic tumorspecimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS associationdomain family protein 1A (RASSF1A) (57%), and O⁶-methylguanine DNAmethylatransferase (MGMT) (34%), and death-associated protein kinase(DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK wassignificantly lower in primary melanomas (n=20) compared to metastaticmelanomas. However, hypermethylation of RAR-β2 was 70% in both primaryand metastatic melanomas. Cell lines had hypermethylation profilessimilar to those of metastatic melanomas. The analysis of these fourmarkers of metastatic tumors demonstrated that 97% had 1 gene(s) and 59%had 2 genes hypermethylated, respectively. The methylation of genes wasverified by bisulfite sequencing. The mRNA transcripts could bere-expressed in melanoma cell lines having hypermethylated genesfollowing treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis ofmelanoma patients' plasma (preoperative blood; n=31) demonstratedcirculating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at leastone of the markers in 29% of the patients. Our findings indicate thatthe incidence of TSG hypermethylation increases during tumorprogression. Methylation of TSG may play a significant role in cutaneousmelanoma progression.

Cancer cells almost invariably undergo loss of genetic material (DNA)when compared to normal cells. This deletion of genetic material whichalmost all, if not all, varieties of cancer undergo is referred to as“loss of heterozygosity” (LOH). The loss of genetic material from cancercells can result in the selective loss of one of two or more alleles ofa gene vital for cell viability or cell growth at a particular locus onthe chromosome. All genes, except those of the two sex chromosomes,exist in duplicate in human cells, with one copy of each gene (allele)found at the same place (locus) on each of the paired chromosomes. Eachchromosome pair thus contains two alleles for any gene, one from eachparent. This redundancy of allelic gene pairs on duplicate chromosomesprovides a safety system. If a single allele of any pair is defective orabsent, the surviving allele will continue to produce the coded protein.

Due to the genetic heterogeneity or DNA polymorphism, many of the pairedalleles of genes differ from one another. When the two alleles areidentical, the individual is said to be homozygous for that pair ofalleles at that particular locus. Alternatively, when the two allelesare different, the individual is heterozygous at that locus. Typically,both alleles are transcribed and ultimately translated into eitheridentical proteins in the homozygous case or different proteins in theheterozygous case. If one of a pair of heterozygous alleles is lost dueto deletion of DNA from one of the paired chromosomes, only theremaining allele will be expressed and the affected cells will befunctionally homozygous. This situation is termed as “loss ofheterozygosity” (LOH) or reduction to homozygosity. Following this lossof an allele from a heterozygous cell, the protein or gene productthereafter expressed will be homogeneous because all of the protein willbe encoded by the single remaining allele. The cell becomes effectivelyhomozygous at the gene locus where the deletion occurred. Almost all, ifnot all, cancer cells undergo LOH at some chromosomal regions.

Through the use of DNA probes, DNA from an individual's normal cells canbe compared with DNA extracted from the same individual's tumor cellsand LOH can be identified using experimental techniques well known inthe art. Alternatively, LOH can be assayed by demonstrating twopolymorphic forms of a protein in normal heterozygous cells, and onlyone form in cancer cells where the deletion of an allele has occurred.See, for example, Lasko et al, 1991, Annu. Rev. Genet. 25:281-314.

Recent advances in molecular biology have revealed that genesis andprogression of tumors follow an accumulation of multiple geneticalterations, including inactivation of tumor suppressor genes and/oractivation of proto-oncogenes. There are over 40 known proto-oncogenesand suppressor genes to date, which fall into various categoriesdepending on their functional characteristics. These include, growthfactors and growth factor receptors, messengers of intracellular signaltransduction pathways, for example, between the cytoplasm and thenucleus, and regulatory proteins influencing gene expression and DNAreplication. Frequent LOH on specific chromosomal regions has beenreported in many kinds of malignancies, which indicates the existence ofputative tumor suppresser genes or tumor-related genes on or near theseloci. LOH analysis is a powerful tool to search for a tumor suppressergene by narrowing and identifying the region where a putative geneexists. By now, numerous LOH analyses, combined with genetic linkageanalysis on pedigrees of familial cancer (Vogelstein et al 1988; Fearonet al 1990; and Friend et al 1986) or homozygous deletion analyses (Callet al 1990; Kinzler et al 1991; and Baker 1989) have identified manykinds of candidate tumor suppressor or tumor-related genes. Also,because allelic losses on specific chromosomal regions are the mostcommon genetic alterations observed in a variety of malignancies,microsatellite analysis has been applied to detect DNA of cancer cellsin specimens from body fluids, such as sputum for lung cancer and urinefor bladder cancer (Rouleau et al 1993 and Latif et al 1993). Moreover,it has been established that markedly increased concentrations ofsoluble DNA are present in plasma of individuals with cancer and someother diseases, indicating that cell free serum or plasma can be usedfor detecting cancer DNA with microsatellite abnormalities (Kamp et al1994 and Steck et al 1997). Two groups have reported microsatellitealterations in plasma or serum of a limited number of patients withsmall cell lung cancer or head and neck cancer (Hahn et al 1996 andMiozzo et al 1996).

Recent developments in cancer therapeutics have demonstrated the needfor more sensitive staging and monitoring procedures to ensureinitiation of appropriate treatment, to define the end points of therapyand to develop and evaluate novel treatment modalities and strategies.In the management of cancer patients, the choice of appropriate initialtreatment depends on accurate assessment of the stage of the disease.Patients with limited or regional disease generally have a betterprognosis and are treated differently than patients who have distantmetastases (Minna et al 1989). However, conventional techniques todetect these metastases are not very sensitive, and these patients areoften not cured by primary tumor resection because they have metastasesthat are not identified by standard methods during preoperative staging.Thus, more sensitive methods to detect metastases in other types ofcarcinomas would identify patients who will not be cured by localtherapeutic measures, for whom effective systemic therapies would bemore appropriate.

The strategy of the present invention is to utilize genetic differencesbetween normal and cancer cells for diagnosis and monitoring of cancerpatients. Many genes coding for proteins or other factors vital to cellsurvival and growth that are lost, can be identified through LOHanalysis of microsatellite, single nucleotide polymorphism (SNP) loci incancer cells and mapped to specific chromosomal regions. Gene expressionmay be suppressed due to hypermethylation in the promoter region ormutation in the gene. In melanoma, mutations of several already-knowntumor suppresser genes such as p53 gene, neurofibromatosis 1 (NF1) gene,and NF2 gene have been reported at a low frequency and deletions and/ormutations of the cyclin dependent kinase 4 (CDK4) inhibitor gene, whichis a responsible tumor suppresser gene for a familial melanoma, havebeen thought to be important genetic changes in tumor development(Miozzo et al., 1996, Cancer Research 56:2285-2288). In addition to thelocus of CDK4 inhibitor gene (9p21), frequent chromosomal deletions havebeen reported on 1p36, 3p25, 6q22-q26, 10q24-q26, and 11q23. (Mao et al1996; Stroun et al 1987; Chen et al, Nat Medicine 1996; and Nawroz et al1996). An efficient method of testing DNA microsatellite or SNP loci forLOH, hypermethylation in the promoter region of a gene, and mutations ina gene allows early diagnosis of melanoma patients and monitoring of theprogression of the disease as well as effectiveness of the therapeuticregimen.

A cellular DNA can be obtained from a sample of a biological fluid bydeproteinizing the sample and extracting DNA according to the procedureswell known in the art. Examples of biological fluids include urine,blood plasma or serum, sputum, cerebral spinal fluid, peritoneal fluid,ascites fluid, saliva, stools, and bone marrow plasma. The DNA to betested may be a fraction of a larger molecule or can be presentinitially as a discrete molecule. Where the test DNA contains twostrands, it may be necessary to separate the strands of the nucleic acidbefore it can be used, e.g., as a template for amplification. Strandseparation can be effected either as a separate step or simultaneouslywith synthesis of primer extension products. This strand separation canbe accomplished using various suitable denaturing conditions, includingphysical, chemical, or enzymatic means. If the nucleic acid is singlestranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates. The product willbe complementary to the single-stranded nucleic acid and will hybridizewith a single-stranded nucleic acid to form a duplex of unequal lengthstrands that may then be separated into single strands to produce twosingle separated complementary strands.

A DNA marker refers to a DNA sequence (e.g., a microsatellite or SNPlocus, a promoter region, or a gene sequence) associated with a specificbiological event (e.g., presence or absence of a gene, hypermethylationof a promoter, mutation in a gene, expression of a gene, and occurrenceof a disease). Microsatellites are short repetitive sequences of DNAwidely distributed in the human genome. Somatic alterations in therepeat length of such microsatellites have been shown to represent acharacteristic feature of tumors. SNP is a common nucleotide variant inDNA at a single site. Each individual has many single nucleotidepolymorphisms that together create a unique DNA sequence. These markerscan be tested either independently or in combination with each other.

Detection of a DNA marker can be accomplished by a number of means wellknown in the art. One means of detecting a DNA marker is by digesting atest DNA sample with a restriction endonuclease. Restrictionendonucleases are well known in the art for their ability to cleave DNAat specific sequences, and thus generate a discrete set of DNA fragmentsfrom each DNA sample. The restriction fragments of each DNA sample canbe separated by any means known in the art. For example, agarose orpolyacrylamide gel electrophoresis can be used to electrophoreticallyseparate fragments according to physical properties such as size. Therestriction fragments can be hybridized to nucleic acid probes whichdetect restriction fragment length polymorphisms (RFLP). There arevarious hybridization techniques known in the art, including both liquidand solid phase techniques. One particularly useful method employstransferring the separated fragments from an electrophoretic gel matrixto a solid support such as nylon or filter paper so that the fragmentsretain the relative orientation which they had on the electrophoreticgel matrix. The hybrid duplexes can be detected by any means known inthe art, for example, by autoradiography if the nucleic acid probes havebeen radioactively labeled. Other labeling and detection means are wellknown in the art and may be used accordingly.

An alternative means for detecting a DNA marker is by using PCR(polymerase chain reaction; see, e.g., U.S. Pat. Nos. 4,683,195,4,683,202, and 4,683,194). This method allows amplification of discreteregions of DNA containing microsatellite sequences. Amplification isaccomplished by annealing, i.e., hybridizing a pair of single strandedprimers, usually comprising DNA, to a target DNA. The primers embraceoligonucleotides of sufficient length and appropriate sequence so as toprovide specific initiation of polymerization of a significant number ofnucleic acid molecules containing the target nucleic acid. In thismanner, it is possible to selectively amplify the specific targetnucleic acid sequence containing the nucleic acid of interest. Morespecifically, the primers are designed to be substantially complementaryto each strand of target nucleotide sequence to be amplified.Substantially complementary means that the primers must be sufficientlycomplementary to hybridize with their respective strands (i.e., with theflanking sequences) under conditions which allow amplification of thenucleotide sequence to occur. The primer is preferably single strandedfor maximum efficiency in amplification but may be double-stranded. Ifdouble-stranded, the primer is first treated to separate its strandsbefore being used to prepare extension products. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of theinducing agent for polymerization. The exact length of a primer willdepend on many factors, including temperature, buffer, and nucleotidecomposition. The oligonucleotide primers for use in the presentinvention may be prepared using any suitable method, such asconventional phosphotriester and phosphodiester methods or automatedembodiments thereof. In one such automated embodiment,diethylphosphoramidites are used as starting materials and may besynthesized as described by Beaucage et al. One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The primers are annealed to opposite strands of the DNAsequence containing a DNA marker, such that they prime DNA synthesis inopposite but convergent directions on a chromosome. Amplification of theregion containing the DNA marker is accomplished by repeated cycles ofDNA synthesis. Experimental conditions conducive to synthesis includethe presence of nucleoside triphosphates and an agent forpolymerization, such as DNA polymerase, and a suitable temperature andpH. Preferably, the DNA polymerase is Taq polymerase which is relativelyheat insensitive. The amplification procedure includes a specifiednumber of cycles of amplification in a DNA thermal cycler. After aninitial denaturation period of 5 minutes, each amplification cyclepreferably includes a denaturation period of about 1 minute at 95° C.,primer annealing for about 2 minutes at 58° C., and an extension at 72°C. for approximately 1 minute. Following the amplification, aliquots ofamplified DNA from the PCR can be analyzed by techniques such aselectrophoresis through agarose gel using ethidium bromide staining.Improved sensitivity may be attained by using labeled primers andsubsequently identifying the amplified product by detectingradioactivity or chemiluminescense on film.

In a preferred embodiment, the assay involves labeling of the PCRprimers with multiple types of chromophore dyes. In another embodiment,the PCR primers are labeled with an atom or inorganic radical, mostcommonly using radionuclides, but also perhaps heavy metals. Radioactivelabels include ³²P, ¹²⁵I, ³H, ¹⁴C, or any radioactive label whichprovides for an adequate signal and has sufficient half-life. Otherlabels include ligands, which can serve as a specific binding pairmember for a labeled ligand, and the like.

Another object of the invention is to provide a method of detecting DNAmarkers in biological fluids, wherein the presence of LOH,hypermethylation, or mutation is associated with the occurrence ofcancer. This method represents a significant advance over suchtechniques as tissue biopsy by providing a non-invasive, rapid, andaccurate method for detecting DNA markers associated with cancer. Thus,the present invention provides a method which can be used to screenhigh-risk populations and to monitor high risk patients undergoingchemoprevention, chemotherapy, immunotherapy, surgical procedure, orother treatment.

According to the method of the present invention, DNA is isolated from abiological fluid of a patient. For comparison, a control DNA sample maybe prepared, for example, from a non-neoplastic tissue from the samepatient, or from a biological fluid or tissue from a normal person. Itis desirable that the alleles used in the allelotype loss analysis bethose for which the subject is heterozygous. Determination ofheterozygosity is well within the skill of the art. Loss of an allele isultimately determined by comparing the pattern of bands corresponding tothe allele in the control sample to the test sample and noting the size,number of bands, or level of amplification of signal of individualbands. For example, LOH may be defined when one allele showed more thana threshold degree (e.g., ≧50%) reduction of peak intensity for serumDNA as compared to the corresponding allele identified in the controlDNA. Methods of detecting hypermethylation of DNA (see Examples below)and mutations in a gene are well known in the art.

This invention also provides a logistically practical assay to monitorthe genetic changes during cancer progression. The events of tumorprogression are dynamic and the genetic changes that concurrently occuralso are very dynamic and complex. The most significant advantage ofthis approach compared to other approaches is the ability to monitordisease progression and genetic changes without assessing the tumor.This is particularly important during early phases of distant diseasespread, in which subclinical disease is undetectable by conventionalimaging techniques. In addition, in advance stage diseases or inoperablesites in which tumor tissue is very difficult or impossible to obtainfor genetic analysis, the present invention provides an alternative forassessing LOH, DNA hypermethylation, and gene mutation.

Because the methods described above require only DNA extraction frombodily fluid such as blood, it can be performed at any time andrepeatedly on a single patient. Blood can be taken and monitored forLOH, DNA hypermethylation, and gene mutation before or after surgery;before, during, and after treatment, such as chemotherapy, radiationtherapy, gene therapy or immunotherapy; or during follow-up examinationafter treatment for disease progression, stability, or recurrence. Themethod of the present invention also may be used to detect subclinicaldisease presence or recurrence with a DNA marker specific for thatpatient since DNA markers are specific to an individual patient's tumor.The method also can detect if multiple metastases may be present usingtumor specific DNA markers.

Further, the invention provides predictive measures of response tocancer therapies and mortality. The method comprises providing a samplefrom the subject and detecting one or more DNA markers in the sample,wherein the status of the DNA markers are indicative of response tocancer therapies and mortality. More specifically, the inventionprovides a method of predicting the probability of survival of a subjectsuffering from a cancer. For example, if LOH, DNA hypermethylationand/or gene mutation occur in a cancer patient, the patient is expectedto have a low probability of survival.

LOH, DNA hypermethylation and gene mutation can also be detected in atissue sample (e.g., a tumor sample). For a tumor sample, if anon-neoplastic tissue is used as a control sample, it can be of the sametype as the neoplastic tissue or from a different organ source. It isdesirable that the neoplastic tissue contains primarily neoplastic cellsand that normal cells be separated from the neoplastic tissue. Ways forseparating cancerous from non-cancerous cells are known in the art andinclude, for example, microdissection of tumor cells from normal cellsof tissues, DNA isolation from paraffin-embedded sections and cryostatsections, as well as flow cytometry to separate aneuploid cells fromdiploid cells. DNA can also be isolated from tissues preserved inparaffin. Separations based on cell size or density may also be used.Once the tissues have been microdissected, DNA can be isolated from thetissue using any means known in the art. Frozen tissues can be minced orhomogenized and then the resulting cells can be lysed using a mixture ofenzyme and detergent, see, for example, Maniatis, Molecular Cloning, aLaboratory Manual, Cold Spring Harbor Laboratory, 1982. The nucleicacids can be extracted using standard techniques such as phenol andchloroform extraction, and ethanol precipitation.

It is another object of the invention to provide kits and packagedproducts for diagnosing, staging and monitoring cancer patients. Such akit or product usually contains a set of reagents for detecting LOH, DNAhypermethylation, and gene mutation. For example, a kit or product mayinclude nucleic acid probes for specified alleles for which the patientis homozygous or heterozygous to detect LOH in these specified alleles.This provides a measure of the extent of genetic change in a neoplastictissue or a biological fluid which can be correlated with a diagnosis orprognosis. In one specific embodiment, the presence or absence of aspecific allele or combination of alleles is tested by amplification ofregions of the DNA markers using pairs of primers which bracket specificregions of the DNA markers on specific chromosome arms containing repeatsequences with polymorphism. Preferably, the assay uses fluorescentlabeling of DNA with multiple types of chromophores. However,radioactive and other labeling techniques known in the art also may beused. Optionally, the kit or product may include a container, and aninsert associated with the container. The insert may be a label or aninstruction sheet with the information as to, e.g., what sample to useand what the indication is if LOH, DNA hypermethylation or gene mutationis detected.

The kit or product may comprise a carrier means being compartmentalizedto receive in close confinement one or more container means such asvials, tubes, and the like, each of the container means comprising oneof the separate elements to be used for detecting DNA markers. Suchelements include a labeled primer pair for amplifying a DNA marker. Theproduct also may include a DNA polymerase for amplifying the target DNA,appropriate amplification buffers and deoxyribonucleoside triphosphates.The nucleic acids in the product may be provided in solution orlyophilized form. Preferably, the nucleic acids will be sterile anddevoid of nucleases to maximize shelf-life.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLES Example 1 Identification of Circulating Tumor-AssociatedEpigenetic Alterations in the Bone Marrow from Breast Cancer PatientsUsing a Hypermethylation Gene Panel Introduction

A variety of genetic alterations including microsatellite instability,allelic loss, and mutation have been described in primary breastcancers. These events result in loss of gene function and have beenimplicated in tumor development and progression. Clinical tools (i.e.,radiographic) used to detect breast cancer progression have been limitedparticularly in the era of earlier disease diagnosis. The most sensitivemethod for the identification of breast cancer progression at the timeof patient diagnosis is histopathologic lymph node evaluation. However20-30% of node-negative breast cancer patients will develop recurrentdisease within 10 years (Fisher et al 1989 and Rosen et al 1989).Therefore, recurrence may be considered a consequence of occultmetastasis not detected at the time of patient diagnosis and treatment.The most frequent site of breast cancer metastasis is bone (Goldhirschet al 1988). Identification of patients at increased risk for systemicmetastasis may improve prognostic staging and provide selection foradditional therapy that may have a significant impact on diseaseoutcome. Assessment of body fluids for circulating tumor cells usingmicroscopy has shown poor results (Molino et al 1991). This technique islabor-intensive, insensitive and subjective. Furthermore, the rapidcirculation and turbulent environment of blood may contribute to the lowyield. In contrast, detection of occult tumor cells in BM usingimmunocytokeratins has been associated with the subsequent developmentof systemic metastasis and shows promise marker of a poorer prognosticoutcome (Braun et al, N Engl J Med 2000). Regardless, identification ofa few tumor cells among a background of one million normal BM cells canbe difficult and tedious. Automated techniques such as RT-PCR canfacilitate identification with improved sensitivity but may havediminished specificity as tumor cell specific mRNA markers are uncommonand expression levels may vary substantially affecting results.

Recently cell-free DNA has been identified in the serum and plasma frompatients with various cancers (Sidransky et al 1997). These circulatingnucleic acids have demonstrated similar genetic alterations andcharacteristics as those found in the primary tumor. Their presence inblood can be readily identified using common PCR techniques and appearto be elevated during disease progression (Silva et al 1999; Taback etal 2001; Muller et al 2003; and Silva et al 2003).

Alternatively, promoter region hypermethylation has been described as acommon genetic abnormality occurring in various cancers. Aberrantmethylation of CpG islands in promoter regions of putativetumor-suppressor and related genes resulting in their silencing has beenimplicated in oncogenesis. Identification of these additional geneticevents may offer a more accurate molecular portrait accounting for atumor's metastatic potential and provide unique tumor-specific surrogatemarkers for monitoring occult disease progression. Methylation-specificreal-time PCR provides a highly sensitive DNA based assay for thedetection of methylated alleles associated with breast cancer (Estelleret al, Cancer Res 2001).

Because BM is the most common site for systemic relapse following breastcancer diagnosis, we attempted to determine whether BM aspirate plasmacould provide a viable source to detect tumor-specific epigeneticalterations associated with systemic metastasis from early stage breastcancer patients.

Methods and Materials

Surgical Specimens and DNA Isolation. BM aspirates were collectedprospectively in 4.5 ml sodium citrate tubes (Becton Dickinson, FranklinLakes, N.J.) through bilateral anterior iliac approach from 33consecutive patients as follows: 17 American Joint Committee on Cancer(AJCC) stage I patients, 14 AJCC stage II patients, and 2 AJCC stage IIIpatients; undergoing surgical resection of their primary breast cancerat the Saint John's Health Center/John Wayne Cancer Institute. Inaddition, BM aspirates were obtained from five healthy female volunteerdonors to serve as controls. Institutional Review Board approved consentforms were signed by all patients prior to participation in the study.BM was drawn and (cell-free supernatant) plasma was separated, filteredand cryopreserved as previously described (Taback et al 2003) Inaddition, match-paired peripheral venous blood was drawn pre-operativelyand DNA was extracted from one ml of both peripheral blood serum and BMaspirate plasma using QIAamp extraction kit (Qiagen, Valencia, Calif.)as previously described (23).

To determine the correlation of gene hypermethylation found in theprimary breast tumor, DNA was isolated from ten 10 μm sections cut fromparaffin-embedded tissue blocks. Samples were deparaffinized,microdissected from normal tissue using laser capture microscopy(Arcturus, Mountain View, Calif.) and incubated in lysis buffer andproteinase K at 37° C. overnight as described previously.

Gene hypermethylation in BM of tumor DNA was analyzed as describedbelow. Additionally, each BM aspirate was assessed for the presence ofoccult tumor cells by standard histologic staining methods usinghematoxylin and eosin (H&E).

Gene Hypermethylation Markers and MSP. Sodium bisulfite modification wasperformed on 1 ug of genomic DNA as previously described (Hoon et al2004). Primer sets were used for the detection of four genes frequentlyhypermethylated in breast cancer: RAS association domain family protein1 A protein (RASSF1A), adenomatous polyposis coli (APC), retinoic acidbinding receptor-β2 (RAR-β2) and MGMT. In addition, MYOD was assessed asan internal control to confirm DNA presence in the final reaction. MSPwas performed with an initial incubation for 15 min at 95° C. followedby 35 cycles (40 cycles for BM and plasma aspirate samples) ofdenaturation at 94° C. for 30s, annealing at 50-56° C., and extensionfor 90 s at 72° C., followed by a final extension step of 72° C. for 5min. For each MSP reaction, normal donor lymphocyte DNA served as anegative control, SssI treated lymphocyte served as a positive control,and water served as a control for contamination.

Clinical and pathologic data was obtained from John Wayne CancerInstitute's Breast Tumor Computer Database. Chi-Square and Wilcoxon RankSum tests were performed for statistical evaluation for the associationof BM methylation status and known prognostic parameters in breastcancer.

Results

Circulating tumor DNA containing gene promoter hypermethylation for anyone marker was identified in the BM of 7 (21%) of 33 patients. The mostfrequently detected hypermethylated gene marker was RASSF1A occurring in5 (15%) patients BM, followed by MGMT in 2 (6%) patients, RAR-β2 and APCin 1 (3%) patient each Table 1. Five patients demonstrated onehypermethylated gene in their BM, whereas two patients had twohypermethylated genes identified and in twenty-six patients nohypermethylated DNA sequences could be detected for any of the genesassessed. No hypermethylation was detected in the BM from five healthyfemale donors.

TABLE 1 Frequency of gene hypermethylation in patient's serum and bonemarrow Frequency in Patients' Body Fluid (n = 33) Marker Serum BoneMarrow RASSF1A  5 (15%)  7 (21%) MGMT 2 (6%) 2 (6%) RARβ 1 (3%) 2 (6%)APC 1 (3%) 0There was an increased association between the presence of genehypermethylation detected in the BM and advanced disease stage. Three(18%) of 17 AJCC stage I patients demonstrated hypermethylated DNA forat least one marker in their BM, in contrast to 3 (21%) of 14 AJCC stageII patients, and 1 (50%) of 2 AJCC stage III patients (Table 2).

TABLE 2 Gene hypermethylation detection in breast cancer patient's serumand bone marrow according to AJCC stage Patients with hypermethylationAJCC Stage Serum Bone Marrow I 4 3 (n = 17) (24%) (18%) II 4 3 (n = 14)(29%) (21%) III 1 1 (n = 2) (50%) (50%)

Hypermethylation was detected in paired peripheral blood serum in 9(27%) of 33 patients. Again RASSF1A was most frequently identifiedoccurring in 7 (21%) patients serum samples followed by RAR-β2 (6%) andMGMT (6%) (Table 1). Eight patients demonstrated hypermethylation inserum for one gene and one patient for 3 genes. Similarly, there was anincreased association between the presence of gene hypermethylation inserum and advanced AJCC stage. Hypermethylation for any one gene wasidentified in 4 (24%) of 17 AJCC stage I patients serum, whereas 4 (29%)of 14 AJCC stage II patients, and 1 (50%) of 2 AJCC stage III patientshad these findings (Table 2).

Twelve clinicopathologic prognostic factors were assessed forcorrelation with BM methylation status: patient age, histologic tumortype, size, grade, Bloom-Richardson score, lymph node involvement, AJCCstage, receptor status (estrogen (ER), progesterone (PR), HER2), Ki-67and p53 status. A trend towards increased circulating methylated DNA inserum from patients with PR negative tumors was identified: 5 (50%) of10 patients as compared to 4 (18%) of 23 patients with PR positivetumors. In multivariate analysis, patients with PR positive tumors wereless likely to have methylation markers in their BM and serum, oddsratio 0.04, 95% CI: 0.00-0.82 (p<0.04). However, due to the small samplesize, no other correlations were identified with BM or serum methylationstatus.

Concordance between the presence of serum and/or BM hypermethylationstatus among patients is shown in Table 3. However, identification ofthe same gene hypermethylated between the BM and serum occurred in only2 patients, with one additional patient having the same genehypermethylation profile in BM for two of the three genes detected inserum.

TABLE 3 Hypermethylation status: concordance between patient's serum andbone marrow Serum Yes No Bone Marrow Yes 3 4 No 6 20 Yes: presence ofhypermethylation detected for any one gene No: absence ofhypermethylation detected for any genes

To determine whether a correlation existed between the genehypermethylation detected in patients BM and their primary tumor, DNAwas isolated from primary tumors and evaluated with the samehypermethylation markers. Of 13 patients with BM and/or serum positivefor gene hypermethylation, 8 had primary tumor blocks available forassessment. In all eight patients, the hypermethylated gene(s)identified in the BM/serum was also hypermethylated respectively in theprimary tumor.

Conventional histologic analysis of all specimens using standard H&Estaining did not demonstrate occult tumor cells in any of the BMsamples.

Discussion

Advances in breast imaging modalities and greater awareness for earlydetection has resulted in a dramatic increase in the number of smallerbreast cancer diagnosed. Concurrently, these tumors are less likely tobe associated with readily identified metastasis. However, patients withsmall primary tumors are not exempt from developing recurrent disease,and these relapses are most likely a consequence of occult metastasispresent at the time of initial diagnosis and treatment. Thus, improvedmethods are needed to detect submicroscopic disease which can identifypatients at increased risk for recurrence sooner in their treatmentcourse. Additionally, techniques that detect occult metastasis maybetter stratify those patients with subclinical disease that may benefitfrom adjuvant therapy while more accurately recognizing patients who donot require additional treatment.

MSP provides a highly sensitive and quantitative technique that canidentify 1 methylated allele among a background of 1000 normal alleles(Herman et al 1996) and therefore may prove useful for assessing thepresence of occult disease and increased patient risk. Additionally,this approach allows for the identification of novel aberrantlyhypermethylated genes, which may be associated with breast cancer tumorgrowth and metastasis and thereby distinguish additional potentialtargets for therapy. In this study, we identified tumor-associatedepigenetic alterations circulating in the blood and BM from 4 (18%) of22 early stage breast cancer patients without evidence of lymph nodemetastasis. It is estimated that 20-30% of node-negative patients willdevelop a systemic recurrence by 10 years and younger patients remain atrisk for relapse many decades after diagnosis (Brenner et al 2004). Thuslonger-term follow-up to determine whether these findings are associatedwith disease recurrence in this group of patients will be needed.However, these findings provide a promising potential for analyzingcirculating nucleic acids in body fluids from patients with breastcancer for assessing the earliest stages of disease progression.Recently, investigators have shown DNA methylation in breast cancerpatients serum to correlate with a worse survival (Muller et al 2003).We have previously demonstrated the presence of LOH in the BM frompatients with breast cancer (Taback et al, 2003). These findings aresignificant because bone is the most frequent site of systemicmetastasis. Therefore, performing a comprehensive assessment of apatient's body fluids, particularly the location most common forrelapse, may yield highly informative information, improve riskassessment and allow for a more accurate method for monitoring treatmentresponses earlier in the disease course. Innovative techniques areneeded to detect and characterize subclinical disease progression inthis new era of early breast cancer diagnosis.

Example 2 Distinct Hypermethylation Profile of Primary Breast Cancer isAssociated with Sentinel Lymph Node Metastasis Introduction

Improved access to mammography and increased patient awareness in breastcancer screening have resulted in a dramatic increase in the detectionof early breast cancers (Cady et al 1996; Shapiro 1982; Tabar 1985; andMiller et al 1993). As important, at the time of breast cancerdiagnosis, is the identification of concurrent metastatic disease foraccurate patient staging and therapeutic decision making. Axillary lymphnode dissection (ALND) has provided an invaluable approach to assess forthe presence of tumor cell metastasis, particularly in early diseasestates where standard radiographic imaging is less sensitive. However,ALND can be associated with considerable morbidity including lymphedemaand reduced shoulder mobility (Ivens et al 1992 and Warmuth et al 1998).In addition, ALND often requires general anesthesia, in patienthospitalization and a postoperative drain. Evidence from prospectiverandomized trials has questioned the therapeutic value of routine ALNDin patients without palpable disease (1980 #44; Fisher et al 1985; andCabanes et al 1992).

Sentinel lymph node (SLN) biopsy provides an effective alternativeapproach to the identification of regional nodal metastasis, and isassociated with reduced morbidity when compared to standard ALND(Giuliano et al 2000). This procedure, although less invasive, is notentirely risk-free as it still requires an axillary incision and generalanesthesia, subjects patients to lymphatic mapping reagents and itssuccess is dependent on the skill of the surgeon (Giuliano et al 1999and Borgstein et al 1998). The main advantage of the technique is thatit provides a more cost-effective, less labor-intensive process forfocused detection of metastasis, particularly when assessing for thepresence of occult tumor cells which are more likely to be associatedwith earlier disease states (Giuliano et al 1998). The addition ofimmunohistochemical (IHC) analysis has further improved theiridentification (Turner et al 1999). The clinical implications of thesefindings remain to be conclusively determined by historical reviews andtherefore prospective multicenter studies are currently underway toevaluate their significance (Wilke et al 2003 and Grube et al 2001).Regardless, a greater number of patients with small primary tumors arebeing treated with adjuvant chemotherapy and hormonal therapy. This maybe a result of recent studies demonstrating a survival advantage inbreast cancer patients without lymph node metastasis [, 1998 #62; 1998#133; and Eifel et al 2001). It must be cautioned that patients withlymph node disease derive the greatest benefit and widespreadapplication of such an aggressive approach may not prove necessary forall cases of early stage breast cancers, as only 20-30% of patientswithout histopathologic evidence of lymph node metastasis willsubsequently develop a recurrence (Winchester et al 1991 and Cooper etal 1991). Adjuvant chemotherapy is associated with potential for patienttoxicity, and its added healthcare costs and resources for itsadministration must be considered (Hillner et al 1991 and Smith et al1993). Consequently, improved methods are needed to better identifypatients at increased risk for disease recurrence and systemicmetastasis, which would provide a more appropriate utilization ofpatient care resources.

Breast cancer development is a consequence of a serial accumulation ofgenetic alterations ultimately resulting in the ability of epithelialcells to proliferate uncontrollably, invade tissues and avoid apoptosis.These genetic events lead to gene activation/inactivation through themechanisms of mutation, amplification and deletion (Sidransky et al1997). More recently, it has been shown that different cancersdemonstrate significant CpG island hypermethylation in the promoterregions(s) of specific tumor-suppressor and related genes regulatingcellular function and contributing to their transcription silencing whencompared to normal cells (Baylin et al 2001 and Esteller et al 2001).These epigenetic events have been suggested to play a significant rolein cancer progression (Widschwendter et al 2002).

Despite the descriptive profile studies of various genes hypermethylatedin breast cancer, relatively little is known of their impact on tumordevelopment and progression (Muller et al 2003). Even more important iswhether these tumor genetic aberrations have clinicopathologic utility.In breast cancer where outcome data such as recurrence and survival canonly adequately be obtained after a relatively prolonged follow-upperiod, correlation with established clinical and pathologic prognosticfactors may serve as an interim surrogate (Stearns et al 2003). Becauselymph node metastasis remains the most significant prognostic factor inpatients with early stage breast cancer, we sought to determine whethera hypermethylation marker panel comprising six tumor suppressor andcancer related genes: RAS association domain family protein 1 A protein(RASSF1A), adenomatous polyposis coli (APC), Twist gene of abasic-helix-loop-helix family of transcription factors, E-cadherin(CDH1), glutathione S-transferase pi 1 (GSTP1) and retinoic acid bindingreceptor-β2 (RAR-β2), detected in primary breast tumors could predictthe likelihood of SLN metastasis.

The SLN has been shown to represent the first site of drainage from aprimary breast tumor and is most likely to harbor detectable metastasisin patients with early stage disease. Thus, the characterization of anepigenetic tumor profile that is associated with SLN metastasis wouldnot only provide better insight into the biology of breast cancerprogression by defining those genetic events associated with theearliest of tumor spreading but may also provide prognostic informationfrom primary tumor assessments. In this study, we developed amethylation-specific PCR (MSP) assay to assess archivedparaffin-embedded primary breast tumors for hypermethylation profiles ofknown tumor-suppressor and related genes with clinicopathologiccorrelation.

Methods

Patients. A total of 151 patients were identified from the Breast CancerDatabase at the John Wayne Cancer Institute who underwent surgery fortheir primary breast cancer with SLN biopsy alone or with ALND fromAugust 1992 to May 2001. Two thirds of the patients were postmenopausaland mean patient age was 55 years (range: 27-86 years) with a mean tumorsize of 3.1 cm (range: 0.1-10 cm). Additional primary tumorcharacteristics are listed in Table 4. The study was approved by thejoint Saint John's Health Center/John Wayne Cancer Institute'sinstitutional review board with all patients providing informed writtenconsent.

TABLE 4 Patient characteristics Factors n = 151 Menopausal pre 51 (33.8)post 100 (66.2)  T Stage T1a 1 (0.7) T1b 4 (2.6) T1c 13 (8.6)  T2 118(78.2)  T3 15 (9.9)  N Stage N0 71 (47.0) N1 74 (49.0) N2 6 (4.0) MStage M0 147 (97.4)  M1 4 (2.6) AJCC Stage I 1 (0.7) IIa 86 (57.0) IIb43 (28.5) IIIa 17 (11.3) IV 4 (2.7) Histology Ductal 118 (78.2)  Lobular33 (21.8) Differentiation Well 30 (20.1) Moderate 62 (41.6) Poor 57(38.3) [Unknown]  [2] Invasion No 102 (70.3)  Yes 43 (29.7) [Unknown] [6] SLN status Negative 70 Micro 40 Macro 41

DNA Extraction and MSP. Paraffin-embedded primary tumor specimen blockswere sectioned at 10 μm deparaffinized in 100% xylene, followed by 100%ethanol incubation and stained with hematoxylin and eosin (H&E). Tumortissue was microdissected in comparison to a similarly stained andcover-slipped reference slide cut in sequence from each tissue block.The samples were incubated in buffer containing SDS-proteinase K for 48hr at 50° C. with an additional 1 μg proteinase K added twice withineach 24 hr period. DNA was extracted and bisulfite modification wasperformed using the agarose bead technique as previously described(Spugnardi et al 2003). Briefly, following extraction, DNA wasquantitated using Picogreen (Molecular Probes, Eugene, Oreg.) and 1 μgof genomic DNA was mixed with, 0.3 M NaOH, 2 vols of 2% LMP agarosedissolved in molecular grade water, heated at 80° C. for 10 min and thenadded to 2-3 drops of chilled mineral oil to create an agarose bead.Sodium bisulfite conversion of DNA suspended in the agarose bead wasachieved by adding 2.5 M sodium metabisulfite and 125 mM hydroquinoneand incubating at 50° C. for 14 hr. Subsequently, desulphonation wasperformed by evacuating residual mineral oil and adding 0.2 M NaOH×2 for15 min each, followed by neutralization with ⅕ vol 1 M HCL for 5 min andthen the bead was washed in Tris-EDTA buffer and stored in moleculargrade water at 4° C. until analysis. A panel of six genes was assessedfor their methylation status: RASSF1A, APC, Twist, CDH1, GSTP1 andRAR-β2. MSP was performed on each bead in a 100 μl reaction containing200 μM each of dNTP and AmpliTaq Gold DNA polymerase (Perkin Elmer,Norwalk, Conn.) and 50 pmol of each forward (F) and reverse (R) primerset for methylated (M) and unmethylated (U) sets as follows: RAR-β2, (M)F-GAACGCGAGCGATTCGAGT (SEQ ID NO:1) and R-GACCAATCCAACCGAAACG (SEQ IDNO:2), (U) F-GGATTGGGATGTTGAGAATGT (SEQ ID NO:3) andR-CAACCAATCCAACCAAAACAA (SEQ ID NO:4); CDH1, (M)F-TTAGGTTAGAGGGTTATCGCGT (SEQ ID NO:5) and R-TAACTAAAAATTCACCTACCGAC(SEQ ID NO:6), (U) F-TAATTTTAGGTTAGAGGGTTATTGT (SEQ ID NO:7) andR-CACAACCAATCAACAACACA (SEQ ID NO:8); APC, (M) F-TATTGCGGAGTGCGGGTC (SEQID NO:9) and R-TCGACGAACTCCCGACGA (SEQ ID NO:10), (U)F-GTGTTTTATTGTGGAGTGTGGGTT (SEQ ID NO:11) and R-CCAATCACAAACTCCCAACAA(SEQ ID NO:12); RASSF1A, (M) F-GTGTTAACGCGTTGCGTATC (SEQ ID NO:13) andR-AACCCCGCGAACTAAAAACGA (SEQ ID NO:14), (U) F-TTTGGTTGGAGTGTGTTAATGTG(SEQ ID NO:15) and R-CAAACCCCACAAACTAAAAACAA (SEQ ID NO:16); GSTP1, (M)F-TTCGGGGTGTAGCGGTCGTC (SEQ ID NO:17) and R-GCCCCAATACTAAATCACGACG (SEQID NO:18), (U) F-GATGTTTGGGGTGTAGTGGTTGTT (SEQ ID NO:19) andR-CCACCCCAATACTAAATCACAACA (SEQ ID NO:20); Twist, (M)F-TTTCGGATGGGGTTGTTATCG (SEQ ID NO:21) and R-GACGAACGCGAAACGATTTC (SEQID NO:22), (U) F-TTGGATGGGGTTGTTATTGT (SEQ ID NO:23) andR-ACCTTCCTCCAACAAACACA (SEQ ID NO:24). PCR was carried out afteroptimizing annealing temperatures for each primer set to include 40timed cycles of denaturation at 94° C. for 30 sec, annealing for 30 sec,and extension at 72° C. for 30 sec. Post-MSP product analysis wasperformed using capillary array electrophoresis (CEQ 8000XL GeneticAnalysis System, Beckman Coulter, Fullerton, Calif.) as describedpreviously (Spugnardi et al 2003).

Sequencing analysis. Sixteen primary breast tumor samples were randomlyselected and analyzed by sequencing to validate the accuracy of the MSPassay for individualized genes. Briefly, PCR was performed on bisulfitemodified DNA in 40 μl reactions with forward and reverse primers forspecific genes as previously described (Spugnardi et al 2003) and (Hoonet al 2004). Fifteen μl of post-PCR products were resolved on 2%Tris-borate EDTA-agarose gels and target bands were isolated andpurified using the Qiagen Gel purification kit (Qiagen Inc., Valencia,Calif.). Sequencing reactions were performed with the dye terminatorcycle sequencing kit on the CEQ 8000XL.

Statistical analysis. Descriptive statistics, such as mean, standarddeviation, median, frequency and percentage were used to summarizepatient's characteristics and gene hypermethylation status. T-test (forcontinuous variables) and chi-square test (for categorical variables)were used for comparing clinical factors between tumors demonstratinghypermethylation versus no hypermethylation.

A logistic regression model was developed to investigate the correlationof gene methylation status with lymph node metastasis status while theeffects of clinical factors on node metastasis were taken into account.First, a stepwise procedure was used to select clinical factors thatsignificantly related with lymph node metastasis status. Tumor size andestrogen receptors status (ER) were selected in the model, a stepwiseprocedure was used again to select genes that predict node metastasisstatus. The statistical analysis was carried out using SAS software(SAS, Cary, N.C.) and all tests are two-sided with significant p values≦0.05.

Results

Promoter region CpG hypermethylation was identified in 147 (97%) of 151primary breast tumors when evaluated for any one marker using thefollowing panel of genes: RASSF1A, APC, Twist, E-cadherin, GSTP1 andRAR-β2. The most frequently hypermethylated gene detected was RASSF1Aoccurring in 122 (81%) patients' tumors; this was followed by E-cadherin(53%), APC (49%), Twist (48%), RAR-β2 (24%) and GSTP1 (21%). Forty-five(30%) of 151 tumors demonstrated hypermethylation for three genes, 43(28%) tumors for two genes, 25 (17%) for four genes, 20 (13%) for onegene, 10 (7%) for five genes, and 4 (3%) for all six genes. In only fourpatient's tumors, hypermethylation was not detected for any of the sixgenes assessed. Sequence analysis was performed on 16 primary tumors toverify the hypermethylated or unmethylated status. In all cases, directsequencing of the PCR product correlated with the methylation status asinitially detected by MSP. Ten normal breast tissue samples demonstratedno promoter hypermethylation for any of the genes assessed.

The individual gene hypermethylation status for each patient's tumor wasassessed to determine whether any clinical or pathologic correlationcould be identified for any of the following prognostic parametersassociated with breast cancer: patient's age, menopause status, tumorsize, histology, degree of differentiation, DNA index, the presence oflymphovascular invasion, T stage, nodal involvement, AJCC stage, hormonereceptor (estrogen and progesterone) status and HER2 receptor presence.GSTP1 methylation was significantly more frequent in primary breasttumors demonstrating lymph node metastasis occurring in 22 (28%) of 81patients, as compared to 10 (14%) of 70 patients without evidence oflymph node involvement (p=0.044). Hypermethylation of the CDH1 was morefrequent in primary tumors demonstrating lymphovascular invasion, 31(72%) of 43 patients versus 49 (48%) of 102 patient tumors withoutlymphovascular invasion (p=0.008); those with an infiltrating ductalhistology, 68 (58%) of 118 tumors as compared to infiltrating lobularhistology 12 (36%) of 33 tumors (p=0.03); and in ER negative tumors, 27(73%) of 37 patients' tumors versus 53 (47%) of 114 patients' tumors(p=0.005). In contrast, RASSF1A hypermethylation was more frequentlyassociated with ER positive tumors occurring in 99 (87%) of 114 patientsversus 23 (62%) of 37 patients with ER negative tumors (p<0.001). RAR-β2hypermethylation was more common in HER2 positive than negative tumors,15 (48%) of 31 cases versus 21 (19%) of 112 cases, respectively(p<0.001). No clinical or pathologic correlations were identified forAPC or Twist hypermethylation.

In a similar manner, the combination of hypermethylated genes wasassessed to determine whether there was any predictive correlation. Thepresence of hypermethylation for GSTP1 and/or RAR-β2 was more frequentlyassociated with the presence of lymph node metastasis and HER2 receptorpositive tumors. Thirty-six (44%) of 81 primary tumors withcorresponding lymph node involvement demonstrated hypermethylation forone or both of these markers, whereas this event was only detected in 18(25%) of 70 primaries without lymph node metastasis (p<0.02).Additionally, hypermethylation for either one or both of these genes wasmore often found in HER2 positive breast cancers than those that wereHER2 negative: 19 (61%) of 31 primary tumors versus 33 (30%) of 112primary tumors, respectively (p=0.001).

It has been suggested that the amount of regional lymph node involvementis associated with a worse patient prognosis (Rosen et al 1981; Nasseret al 1993; and Goldstein et al 1999). To determine whetherhypermethylation profiling of the primary tumor was predictive of lymphnode tumor burden, patients were categorized according to the size ofthe SLN metastasis: macro, >2.0 mm (n=41); micro, ≦2.0 mm (n=40); andnone (n=70), absence of tumor identification following H&E and IHCstaining. Among these three groups there was a statistically significantassociation between increasing SLN tumor burden and larger primary tumorsize (p<0.015). Correlation with primary tumor hypermethylation statusfound a greater frequency of GSTP1 hypermethylation associated withmacro-SLN metastasis, 13 (32%) of 41 patients, as compared to thosewithout tumor cells in the SLN, 10 (14%) of 70 patients, p<0.029. RAR-β2hypermethylation was more common in those tumors having macro-SLNmetastasis, 17 (42%) of 41 patients, versus micro-SLN metastasis, 6(15%) of 40 patients, or no SLN metastasis, 13 (19%) of 70 patients,(p<0.009 for each, respectively). Similarly, the presence of eitherGSTP1 hypermethylation, RAR-β2hypermethylation, or both was morefrequently observed in primary tumors having macro-SLN metastasis, 23(56%) of 41 patients, than micro-SLN metastasis, 12 (30%) of 40patients, or no SLN metastasis, 18 (26%) of 70 patients, p values <0.018and 0.002, respectively.

A logistic regression model was developed to investigate the correlationof gene methylation status with SLN tumor status while the effects ofclinical factors on node metastasis were taken into account. Only tumorsize and RAR-β2 gene hypermethylation were significantly associated witha greater risk for a macro-SLN metastasis as compared to micro- or noSLN involvement, odds ratio of 1.5 (95% CI, 1.16 to 1.93; p<0.002) and3.86 (95% CI, 1.65 to 9.00; p<0.002). Similarly, in multivariateanalysis, the presence of either GSTP1 hypermethylation, RAR-β2hypermethylation or both markers in primary tumors correlated with anincreased risk of having a macroscopic SLN metastasis, odds ratio 4.59(95% CI, 2.02 to 10.4; p<0.001). Increasing primary tumor size was alsoassociated with a greater risk for macro-SLN metastasis, odds ratio 1.57(95% CI, 1.21 to 2.05; p<0.001). No clinical, pathologic orhypermethylation gene marker variables could discriminate betweenmicroscopic SLN metastasis and histologically tumor-free SLN.

TABLE 5 Correlation between primary tumor gene hypermethylation markerand patient tumor characteristics Gene hypermethylation and tumorhistopathology P-value GSTP1 Lymph node positive 0.044 CDH1 Lymph nodepositive 0.008 Infiltrating ductal histology 0.03 Estrogen receptorpositive 0.005 RASSF1A Estrogen receptor positive <0.001 RAR-β2 Her2receptor positive <0.001 GSTP1 and/or RAR-β2 Lymph node positive <0.02Her2 receptor positive 0.001

TABLE 6 Correlation between primary tumor gene hypermethylation markerand SLN histology status Gene hypermethylation and SLN histopathologyP-value GSTP1 Macro vs. None <0.015 RAR-β2 Macro vs. Micro <0.009 Macrovs. None <0.009 GSTP1 and/or RAR-β2 Macro vs. Micro <0.018 Macro vs.None <0.002 Macro, metastasis >2 mm; Micro, metastasis ≦2 mm; None, nometastasis detected by H&E and IHC

Discussion

Breast cancer remains the most frequently diagnosed malignancy in women(Jemal et al 2003), the incidence of which continues to rise yearly. Asof lately, many of these detected cases of breast cancer are smaller insize than those reported in previous decades and therefore less likelyto be associated with overt lymph node metastasis (Chu et al 1996).Because prospective randomized trials have not demonstrated a survivaladvantage with up-front ALND, its therapeutic utility in this new era ofbreast cancer diagnosis will only further diminish (Fisher et al 2002).Regardless, at present, tumor status of the axillary lymph nodes is thesingle most important clinically used predictor of patient outcome todate (Mansour et al 1994). Furthermore, lymph node evaluation remains amainstay for disease staging, as a treatment stratification factor, andfor assessing overall patient prognosis.

The more frequent utilization of chemotherapy in patients withnode-negative breast cancer may further contribute diminishing role ofSLN biopsy. In addition there has been a dramatic increase in breastconserving therapy, which entails local radiation. The additionaleffects of these local and systemic therapies on minimal residualdisease in axillary lymph nodes is at present unknown, but may provebeneficial and further reduce the need for lymph node surgery inpatients with early stage breast cancer.

Discrimination of primary tumors based on molecular characteristics mayprove useful for predicting lymph node metastasis, risk of recurrence,and improving our understanding of the etiologic events that promotedisease spreading (Isaacs et al 2001). Advantages of gene-based assaysare their rapidity for assessment, widespread use of currentlyimplemented technology, objectivity of results and requirement of only aminimal amount of sample without imposing excessive demands on stringentcollection and processing techniques. Additionally, DNA assay studiesallow for easy evaluation of available paraffin-embedded tissuespecimens. Finally, DNA-based assays offer an alternative to RNA-basedapproaches, which may be affected by heterogeneity, variations in thelevels of gene expression, and most importantly RNA degradation. Thesefactors have proven problematic in large-scale studies.

This study provides the largest series to date with correlation to knownprognostic factors in breast cancer to determine the role of genepromoter hypermethylation status as a molecular predictor of diseaseprogression. We found GSTP1 methylation to correlate strongly withincreasing tumor size and a greater likelihood of SLN metastasis. Thisfinding is important, as GSTs are a family of enzymes that detoxifyhydrophobic electrophiles, which include carcinogens that have beenimplicated in a variety of cancers (Henderson et al 1998). GSTP1 lossappears to be an early event in the development of prostate cancer andloss of this enzyme may impair cellular defenses leading to increasinggenome instability and cancer progression (Nelson et al 2002). Becausebreast cancer is similarly a hormone mediated malignancy andepidemiologic studies have shown diet and its components as potentialcontributing factors to its development, this same enzyme may becritical in this disease as well (Clavel-Chapelon et al 1997 andKrajinovic et al 2001). Additional studies using large-scale populationswill better identify these risks and characterize the potential impactof gene-environment interactions.

Hypermethylation of RAR-β2 was shown to correlate more frequently withHER2 positive tumor, which is over-expressed in 25-30% of all breastcancers and when identified is associated with a poorer patientprognosis. Retinoids have been shown to inhibit the growth of breastcancer cell lines in culture and breast tumors in animal models [Lacroixet al 1980; Fraker et al 1984; and Gottardis et al 1996). RAR-β2 hasbeen proposed as a tumor suppressor gene and loss of expression has beenfound in variety of tumors as well as premalignant lesions resulting inuncontrolled cellular proliferation (Martinet et al 2000 and Sun et al2002). Detection of RAR-β2 hypermethylation may identify additionaltherapeutic targets of interest in these groups of patients with moreaggressive tumors. The correlation of RAR-β2 with the presence ofmacroscopic SLN metastasis is significant. Tumor burden in the lymphnodes is a significant prognosticator of patient outcome. However, theclinical implication of occult tumor cells in lymph nodes remains acontroversial issue (Dowlatshahi et al 1997 and Cote et al 1999). Wehave demonstrated that patients with SLN micrometastasis (≦2.0 mm) haveequivalent overall survival rate as those without SLN metastasis andboth groups have a better outcome than those with SLN macrometastasis(>2.0 mm) (Hansen et al 2001). Genetic markers that predict for lymphnode metastasis may avoid further surgery in patients with clinicallyinsignificant disease in their axilla and better identify those morelikely to benefit from the addition of systemic therapy. Methylationstatus of the RAR-β2 may identify patients suitable for enrollment intoclinical trials employing retinoids (Lawrence et al 2001 and Singletaryet al 2002).

CDH1 hypermethylation was highly associated with ER negative tumors.E-cadherin is involved in cell-to-cell adhesion and the metastasisprocess. Loss of heterozygosity for this gene with near complete absenceof CDH1 protein expression is highly common for invasive lobular breastcancers, whereas tumors of ductal histology often present with varyinglevels of expression (Asgeirsson et al 2000 and Cleton-Jansen et al2002) Promoter region hypermethylation may provide an alternativemechanism to account for this finding in ductal carcinomas.

Promoter region hypermethylation is a common epigenetic event that hasbeen shown to occur among a variety of different tumor types affectingmultiple genes that regulate cell cycling, signal transduction, genetranscription, angiogenesis, adhesion and metastasis. Hypermethylationprofiling of specific genes in cancers can characterize those geneticalterations associated with tumorigenesis and metastasis (Krassensteinet al 2004). Identification of specific tumor-associated genetic eventscan potentially account for the pathobiology of tumor progression andprovide molecular markers for assessing patient risk, monitoring tumorprogression and predicting response to therapy. In addition, thisapproach allows for the detection of alterations in pathways critical tomaintaining cell integrity, stability, survival and chemoresistancewhich can identify unique patient-specific targets to customizetherapies for improved treatment response. Furthermore, development ofepigenetic therapeutic protocols may prove useful in the future aspreventatives for individuals at high risk for breast cancer. Molecularevents associated with the primary tumor that predict for metastasis andpatient outcome offers the desired opportunity to avoid additionalsurgical interventions for staging and will prove more suitable in thisnew era of earlier cancer detection.

Example 3 Profiling Epigenetic Inactivation of Tumor Suppressor Genes inTumors and Plasma From Cutaneous Melanoma Patients Introduction

Epigenetic events in the form of hypermethylation of TSG promoterregion(s) CpG islands can play a role in the development and progressionof various cancers (Baylin et al 2000; Esteller et al 2001; Jones et al2002; and Sidransky et al 2002). The detection of hypermethylated genesin tumors has become important in assessing the mechanisms of known andcandidate TSG inactivation. Genes can be transcriptionally silenced whentheir promoter region(s) CpG islands are hypermethylated (Jones et al2002). Recent studies have shown this is a significant mechanism wherebyTSG expression is shut off in cancer cells (Baylin et al 2000; Estelleret al 2001; Jones et al 2002; Sidransky et al 2002). Thehypermethylation status of several known or candidate TSG promoterregions has been profiled for a number of cancers (Esteller et al 2002;Harden et al 2003; Jeronimo et al 2001; Jones et al 2002; Lo et al 2001;Pfiefer et al 2002; Rosas et al 2001; Toyooka et al 2003; Widschwendteret al 2002). This epigenetic regulation of TSG can provide a selectiveadvantage for cells undergoing transformation or progressing to a moremalignant phenotype. In the past, considerable effort was devoted tocorrelating known or candidate TSG deletions and mutations to phenotypicproperties. Recent studies have indicated that inactivation of specificTSGs significantly influences tumor promotion and progression incarcinomas (Harden et al 2003; Jeronimo et al 2001; Jones et al 2002; Loet al 2001; Rosas et al 2001; Toyooka et al 2003; and Widschwendter etal 2002).

Most studies on hypermethylation of gene promoter regions have focusedon carcinomas; no major study has addressed hypermethylation of TSG incutaneous melanomas. The genetic mechanisms involved in melanoma tumorprogression are poorly understood. BRAF mutation V599 (Davies et al2002) and 9p21 region chromosome deletions (Fujiwara et al 1999) are themajor genetic aberrations frequently (>40%) found so far in sporadicprimary or metastatic cutaneous melanomas. The frequency of othertumor-related gene mutations or deletions is less than 25% in melanomas.This suggests that there are potential genetic aberrations that have yetto be identified. We recently reported on the frequent hypermethylation(>40%) of RASSF1A in melanoma cell lines and frozen metastatic melanomaspecimens (Spugnardi et al 2003). Although its function remainsuncertain, RASSF1A is considered a strong candidate as a TSG.

To date, there has been no major study profiling hypermethylation ofknown or potential TSGs of cutaneous melanomas. We assessed thehypermethylation status of several known or candidate TSG promoterregions in melanoma cell lines and in frozen and paraffin-embeddedmelanoma tissues. Several major TSG were frequently hypermethylated inprimary tumors and more so in metastatic tumors. Some prominentlymethylated genes in carcinomas were infrequently methylated inmelanomas.

Tumor-related DNA circulates in serum/plasma of patients with melanomaand other types of tumors (Fujiwara et al 1999; Johnson et al 2002;Sidransky et al 2002; and Usadel et al 2002). Studies in melanomapatients have shown that specific microsatellites with loss ofheterozygosity (LOH) on different chromosomes are frequent with diseaseprogression (Fujiwara et al 1999). Recent studies have shown thathypermethylated tumor-related DNA can be detected circulating in blood(Sidransky et al 2002 and Usadel et al 2002). We examined thefeasibility of detecting hypermethylated TSG in plasma of melanomapatients. Circulating DNA of three hypermethylated genes wasdemonstrated in plasma of melanoma patients.

Materials and Methods

Cell Lines and Tissues. Fifteen established melanoma cell lines werecultured in growth medium and prepared for DNA extraction as previouslydescribed (Fujiwara et al 1999). Frozen metastatic melanoma tumorspecimens (n=53) were obtained from 44 patients who underwent electivesurgery at John Wayne Cancer Institute, Saint John's Health Center,Santa Monica, Calif. Frozen tumor-draining lymph nodes (n=10),histopathology tumor negative by immunohistochemistry, were obtainedfrom melanoma patients having elective surgery. Paraffin-embeddedmetastatic tumor tissues (n=33) and primary tumors (n=20) from melanomapatients were obtained from the Division of Surgical Pathology at SaintJohn's Health Center. Paraffin-embedded melanoma and breast cancertumor-draining lymph nodes (n=12) that were histopathology(immunohistochemistry) negative were assessed. For the studies on pairedtumors and plasma from the same patients there were additional 24metastatic tumors assessed; tumors from seven pairs overlapped theinitial 86 metastatic patients assessed. All patients had given signedinformed consent to participate in the studies. Human subjects IRBapproval was obtained for the use of human subjects in this study fromSaint John's Health Center and John Wayne Cancer Institute jointcommittee.

Bisulfite Treatment. DNA was isolated from cell lines and frozen tissuesusing DNAzol Genomic DNA Isolation Reagent (Molecular Research Center,Inc., Cincinnati, Ohio) according to the manufacturer's recommendations.Paraffin-embedded tumor DNA was extracted as previously described(Fujiwara et al 1999). Following extraction, DNA was subjected tosodium-bisulfite modification (Spugnardi et al 2003). One μg of DNA wasdenatured at 100° C. for 10 min and quickly chilled on ice. Sodiumhydroxide (Sigma, St. Louis, Mo.) was then added to a finalconcentration of 0.3 M, and the DNA incubated at 50° C. for 15 min. TheDNA was then mixed with 2 vol 2% LMP agarose (BioWhittaker MolecularApplications, Rockland, Me.) and pipetted into chilled mineral oil,forming a single bead. Four hundred μl of a 5 M bisulfite solution (pH5.0) consisting of 2.5 M sodium metabisulfite and 125 mM hydroquinone(Sigma) was added, and the reaction incubated at 50° C. for 14 h. Themodification was stopped by equilibrating the beads with 0.5 ml 1×Tris-EDTA (TE) (6×15 min) followed by desulphonation using 0.2 M NaOH(500 μl, 2×15 min). The reactions were neutralized using 100 μl (⅕ vol)1 N hydrochloric acid (Sigma). One final TE rinse was followed byequilibration in molecular grade H₂O (2×15 min). The beads were thenused for MSP.

Genomic Sequencing. DNA sequences were amplified by mixing 100 ng ofbisulfite treated melanoma cell line DNA with 100 pmoles of eachrespective primer: MGMT, M1 5′-GGGTTATTTGGTAAATTAAGGTATAGAG-3′ (SEQ IDNO:25) and M2 5′-CACCTAAAAATAAAACAAA AACTACCAC-3′ (SEQ ID NO:26);RASSF1A, R3 5′-GGGAGTTTGAGTTTATTGAGTTG-3′ (SEQ ID NO:27) and R2 5′-CACCTCTACTCATCTATAACCCAAATAC-3′ (SEQ ID NO:28); RAR-β2, RA35′-GTGTGATAGAAGTAGTA GGAAGTGAGTTGT-3′ (SEQ ID NO:29) and RA25′-ACTCCATCAAACTCTACCCCTTTTTTAAC-3′ (SEQ ID NO:30) in a 50 μl reactioncontaining buffer, of dNTP and AmpliTaq gold polymerase (AppliedBiosystems, Foster City, Calif.) at 95° C. for 45 s, 55° C. for 45 s and72° C. for 2 min for 40 cycles. PCR products were gel purified using theQIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.) and sequencedusing an automated DNA sequencer (CEQ 8000XL DNA Analysis System,Beckman Coulter, Fullerton, Calif.) with the respective internal primer:MGMT, M3 5′-GTTGT(c/t)GGAGGATTAGGGT-3′ (SEQ ID NO:31); RASSF1A, R45′-TACCCCTTAACTACCCCTTCC-3′ (SEQ ID NO:32), and RAR-β2, RA45′-AATCATAAATTATAACAAACAAACCAACT-3′ (SEQ ID NO:33).

Fluorescent MSP Analysis. Methylation status was assessed for each geneusing two sets of fluorescent labeled primers specifically designed toamplify methylated or unmethylated DNA sequence. Primer sequences arelisted as methylated sense and antisense followed by unmethylated senseand antisense, with annealing temperatures and PCR product size:TIMP-3,5′-CGTTTCGTTATTTTTTGTTTTCGGTTTC-3′ (SEQ ID NO:34) and5′-CCGAAAACCCCGCCTCG-3′ (SEQ ID NO:35) (59° C., 116 bp)5′-TTTTGTTTTGTTATTTTTTGTTTTTGGTTTT-3′ (SEQ ID NO:36) and 5′-CCCCCAAAAACCCCACCTCA-3′ (SEQ ID NO:37) (59° C., 122 bp) (19); RASSF1A,5′-GTGTTAACGCGTTGCGTATC-3′ (SEQ ID NO:13) and5′-AACCCCGCGAACTAAAAACGA-3′ (SEQ ID NO:14) (60° C., 93 bp),5′-TTTGGTTGGAGTGTGTTAATG TG-3′ (SEQ ID NO:15) and5′-CAAACCCCACAAACTAAAAACAA-3′ (SEQ ID NO:16) (60° C., 105 bp) (11,16);RAR-β2, 5′-GAACGCGAGCGATTCGAGT-3′ (SEQ ID NO:1) and5′-GACCAATCCAACCGAAACG-3′ (SEQ ID NO:2) (59° C., 142 bp),5′-GGATTGGGATGTTGAGAATGT-3′ (SEQ ID NO:3) and5′-CAACCAATCCAACCAAAACAA-3′ (SEQ ID NO:4) (59° C., 158 bp) (Evron et al2001); MGMT, 5′-TTTCGACGTTCGTAGGTTTTC GC-3′ (SEQ ID NO:38) and5′-GCACTCTTCCGAAAACGAAACG-3′ (SEQ ID NO:39) (66° C., 81 bp),5′-TTTGTGTTTTGA TGTTTGTAGGTTTTTGT-3′ (SEQ ID NO:40) and5′-AACTCCACACTCTTCCAAAA ACAAAAC (SEQ ID NO:41) (66° C., 93 bp) (Estelleret al 1999); DAPK 5′-GGATAGTCG GATCGAGTTAACGTC (SEQ ID NO:42) and5′-CCCTCCCAAACGCCGA (SEQ ID NO:43) (64° C., 98 bp), 5′-GGAGGATAGTTGGATTGAGTTAATGTT-3′ (SEQ ID NO:44) and 5′-CAAATCCCTCCCAAACACCAA-3′(SEQ ID NO:45) (64° C., 106 bp) (Goessl et al 2000);GSTP1,5′-TTCGGGGTGTAGCGGTCGTC-3′ (SEQ ID NO:17) and5′-GCCCCAATACTAAATCACGACG-3′ (SEQ ID NO:18) (59° C., 91 bp),5′-GATGTTTGGG GTGTAGTGGTTGTT-3′ (SEQ ID NO:19) and5′-CCACCCCAATACTAAATCACA ACA-3′ (SEQ ID NO:20) (59° C., 97 bp) (Estelleret al 1999 and Zochbauer-Muller at al 2001); p16^(INK4a), 5′-TTATTAGAGGGTGGGGCGGATCGC-3′ (SEQ ID NO:46) and 5′-GACCCGAACCGCGACCGTAA-3′ (SEQ IDNO:47) (65° C., 150 bp), 5′-TTATTAGAGGGTGGGGTGGATTGT-3′ (SEQ ID NO:48)and 5′-CAACCCCAAACCACAACCATAA-3′ (SEQ ID NO:49) (65° C., 151 bp)(21,24); and MYOD1,5′-CCAACTCCAAATCCCCTCTCTAT-3′ (SEQ ID NO:50) and5′-TGATTAATTTAGATTGGGTTTAGAGAAGGA-3′ (SEQ ID NO:51) (60° C., 162 bp)(Eads et al 1999). One hundred ng of bisulfite-modified DNA wassubjected to PCR amplification in a final reaction volume of 20 μlcontaining PCR buffer, 2.5-4.5 mM MgCl₂, dNTPs, 0.3 μM primers, BSA and0.5 U of AmpliTaq gold polymerase (Applied Biosystems). PCR wasperformed with an initial 10 min incubation at 95° C., followed by 40cycles of denaturation at 95° C. for 30 s, annealing for 30 s, andextension at 72° C. for 30 s, and a final seven min hold at 72° C.Sodium-bisulfite modified lymphocytes from healthy donors were used aspositive unmethylated control, Sss/Methylase (New England BioLabs,Beverly, Mass.) treated modified lymphocytes were used as a positivemethylated control, and unmodified lymphocytes were used as a negativecontrol for methylated and unmethylated reactions. PCR products werevisualized using capillary array electrophoresis (CAE; CEQ 8000XL). Theassay was set up in a 96-well microplate format. Multiple PCR productscan be assayed in the same well for comparison. Methylated andunmethylated PCR products from each sample were assessed simultaneouslyby labeling forward primers with a choice of three Beckman CoulterWellRED Phosphoramidite (PA)-linked dyes (Genset Oligos, Boulder,Colo.). Forward methylated specific primer was labeled with D4pa dye andforward unmethylated specific primer was labeled with D2pa dye. One μlof methylated PCR product and one μl of unmethylated PCR product weremixed with 40 μl loading buffer and 0.5 μl dye-labeled size standard(Beckman Coulter Inc). The CAE analysis detects the different dyes anddisplays them in respective colors.

RT-PCR Analysis. Total cellular RNA was extracted from melanoma celllines using TriReagent (Molecular Research Center, Cincinnati, Ohio).RT-PCR was performed as previously described (Takeuchi et al 2003).Briefly, all RT reactions were carried out with oligo-dT priming using 1μg of total RNA. Resulting cDNA was subjected to PCR conditions of 95°C. for 30 s, annealing for 30 s, and 72° C. for 1 min, for 35 cycles forMGMT, RASSF1A and RAR-β2 and 25 cycles for the GAPDH housekeeping-gene.All samples were assessed for presence of GAPDH mRNA. All PCR productswere separated on 2% Tris-borate EDTA agarose gels and stained withethidium bromide as previously described (Spugnardi et al 2003).

Reexpression of MGMT, RAR-β2, RASSF1A genes. Several cell lines weregrown for four days in T75 cm² tissue culture flasks in the presence of0, 3, or 5 μM of 5Aza-dC (Sigma) as previously described (Spugnardi etal 2003). An additional flask of cells was grown in the presence of 5 μM5Aza-dC for four days followed by treatment with 1 μM ATRA (Sigma) for24 h. RNA was isolated and RT-PCR was performed as described above toanalyze for RASSF1A, MGMT and RAR-β2 gene reexpression.

Plasma DNA Isolation and Methylation Analysis. DNA was extracted fromplasma as previously described (Taback et al 2001). Briefly, 500 μl ofplasma was diluted with 0.9 M NaCl₂, SDS, and proteinase K (QIAGEN) andincubated at 50° C. for 3 h. An equal volume of phenol-chloroformisoamyl alcohol (25:24:1) was then added and the sample was vortexedvigorously. After centrifugation at 1000×g for 10 min the aqueous layerwas collected and phenol-chloroform isoamyl alcohol was again added. DNAwas precipitated using pellet paint NF co-precipitant (Novagen, Madison,Wis.) and isopropanol.

Extracted DNA was subjected to sodium bisulfite modification. Briefly,DNA extracted from 500 μl of plasma and supplemented with 1 μg salmonsperm DNA (Sigma) was denatured in 0.3 M NaOH for 15 min at 37° C. Fivehundred fifty μl of a 2.5 M sodium bisulfite/125 mM hydroquione solutionwas then added and samples were incubated under mineral oil in the darkfor 3 h at 60° C. Salts were removed using the Wizard DNA Clean-UpSystem (Promega, Madison, Wis.) and samples were then desulfonated in0.3 M NaOH at 37° C. for 15 min. Modified DNA was precipitated withethanol using Pellet Paint NF (Novagen) as a carrier and thenresuspended in molecular grade H₂O.

The methylation status of the bisulfite-treated DNA was determined usingprimers and probes specifically designed to amplify methylated genepromoter regions. Quantitative RealTime PCR was preformed as previouslydescribed (Takeuchi et al 2003). RealTime PCR reactions were run on theiCycler iQ RealTime thermocycler (Bio-Rad Laboratories, Hercules,Calif.). Analysis involved 25 μL PCR reaction containing sodiumbisulfite treated DNA template, 0.8 μM of each primer, 0.4 μMfluorescence resonance energy transfer probe, AmpliTaq gold polymerase(Applied Biosystems), dNTPs, MgCl₂, BSA, and AmpliTaq PCR buffer.Amplification conditions were 95° C. for 10 min followed by 55 cycles ofdenaturation at 95° C. for 1 min, annealing at 60° C. for MYOD1 andRASSF1A (annealing at 59° C. for RAR-β2 and 66° C. for MGMT) andextension at 72° C. for one min. Each PCR plate contained positivecontrols including melanoma cell lines shown to be methylated for thegene being assessed as well as SssI treated healthy donor lymphocytes.Negative controls included healthy donors plasma DNA and reactions whichcontained no template DNA.

Realtime PCR assay was performed to obtain the approximate number ofmethylated gene copies present in a sample. The internal reference geneMYOD1 was used to amplify sodium bisulfite treated DNA independent ofmethylation status to confirm presence of modified DNA (9). In addition,a standard curve was constructed with serial dilutions of 10¹ to 10⁵copies of the targeted TSG promoter region template. Copy numbers forthe individual samples were established using the standard curve. Primersequences for the realtime PCR were the same as for the CAE analysiswhile the probe sequences were as follows: MYOD,5′-CCCTTCCTATTCCTAAATCCAACCTA-3′ (SEQ ID NO:52); MGMT,5′-CGTTTGCGATTTGGTGAGTGTT TGGG-3′ (SEQ ID NO:53); RASSF1A,5′-CAACTACCGTATAAAATTACACGCGATACCCCG-3′ (SEQ ID NO:54); and RAR-β2,5′-CCGAATACGTTCCGAATCCTACCCCG-3′(SEQ ID NO:55).

Results

Methylation profiling of melanoma cell lines. Initially, seven known orcandidate TSG were assessed for aberrant methylation of CpG promoterregions in melanoma cell lines. Methylation status was analyzed usingMSP and assessed by gel electrophoresis initially to verify specificbands. The assay was converted for automated CAE analysis which provideda more objective detection system for methylation status, allowing bothmethylated and unmethylated MSP products to be assessed simultaneouslyin the same analysis. A comparison of CAE versus gel electrophoresisdemonstrated >95% concordance of results. All subsequent tissue analyseswere performed by CAE. The frequency of promoter hypermethylation in thegenes DAPK, GSTP1, MGMT, p16^(INK4a), RAR-β2, RASSF1A, and TIMP-3 wasassessed by MSP in 15 established melanoma cell lines. The most frequenthypermethylated gene was RASSF1A followed by RAR-β2 and MGMT (Table 7).Overall, 14 (93%) of the lines were hypermethylated for one or more ofthe seven genes. Eight (53%) cell lines had two or more hypermethylatedgenes, and two (13%) cell lines had three hypermethylated genes.Positive controls for methylated gene promoter regions included knownhypermethylated cell lines and bisulfite-modified SssI treated normaldonor PBLs. Negative controls for both methylated and unmethylatedprimer sets included unmodified (wild-type) DNA and reagent controls.Under the conditions used normal (histopathology tumor negative) frozenlymph nodes (n=10) and healthy donor PBLs did not show methylation forany gene except DAPK, which was positive in PBLs from two healthydonors.

TABLE 7 Methylation of Gene Promoter CpG Islands in Melanoma Cell LinesMelanoma cell lines RAR-β2 RASSF1A MGMT DAPK GSTP1 TIMP3 p16^(INK4a) MA− + − − − − − MB − − − − − − − MC − + − − − − − MD + − + − − − − ME + +− − − − − MF − + − − − − − MG − + − − − − − MH + + + − − − − MI + + − −− − − MJ + + − − − − − MK + + + − − − − ML + − − − − − − MM + + − − − −− MN − + − − − − − MO − + + − − − − Total 8/15 12/15 4/15 0/15 0/15 0/150/15 (53%) (80%) (27%) (0%) (0%) (0%) (0%)

Methylation profiling of melanoma tumors. We next assessed 53 frozenmetastatic melanoma tumor tissues obtained from 44 AJCC stage III/IVmelanoma patients (FIG. 1, Table 8). Hypermethylation was detected inone or more genes of 51 (96%) tumors, two or more genes of 34 (64%)tumors, and three or more genes of 13 (25%) tumors (Table 9). The fourmost frequently hypermethylated genes were RAR-β2, RASSF1A, MGMT, andDAPK, respectively (Table 8). The other three genes were hypermethylatedin less than 10% of the tumors.

TABLE 8 Detection of Hypermethylated Genes in Melanoma Tumors MGMTRAR-β2 RASSF1A DAPK Primary tumors Paraffin n = 20 2 (10) 14 (70)  3(15) 0 (0) Metastatic tumors Frozen n = 53 20 (38) 38 (72) 31 (58) 13(25) Paraffin n = 33  9 (27) 22 (67) 18 (55) 3 (9) Total n = 86 29 (34)60 (70) 49 (57) 16 (19) ( ) percentage

TABLE 9 Frequency of Hypermethylated Genes in Melanoma Tumors Genes^(a)Frozen n = 0 Paraffin n = 20 Total Primary tumors 0 N/A  5 (25)  5 (25)N = 20 ≧1 N/A 15 (75) 15 (75) ≧2 N/A  4 (20)  4 (20) ≧3 N/A 0 (0) 0 (0)Genes* Frozen n = 53 Paraffin n = 33 Total Metastatic 0 2 (4) 1 (3) 3(3) tumors ≧1 51 (96) 32 (97) 83 (97) N = 86 ≧2 34 (64) 17 (52) 51 (59)≧3 13 (25)  3 (11) 16 (19) 4 4 (8) 0 (0) 4 (5) ( ) percentage ^(a)Genesassessed were: RAR-β2, MGMT, DAPK, and RASSF1A.

Because molecular assessment of frozen tumor tissues is hampered byavailability, size of lesion surgically removed, logistics of tissueprocurement, not knowing the level of contamination with hemopoieticcell infiltrates and normal cells, the MSP assay was adapted foranalysis of paraffin-embedded tumor tissue. We focused on the four genesmost frequently hypermethylated in frozen melanomas: RAR-β2, RASSF1A,MGMT, and DAPK. We initially assessed 11 paired frozen andparaffin-embedded melanomas to verify that the sensitivity of the assaywas equivalent for both types of tissues. There was 100% concordance forall four markers between frozen and paraffin-embedded melanomas.

Primary melanomas were assessed only from paraffin-embedded tissuesopposed to fresh frozen tissues due to the lesion size, and logistics inprocurement for pathology analysis. Paraffin-embedded primary melanomas(n=20) and metastatic melanomas (n=33) were assessed (FIG. 1, Table 8).The most frequently hypermethylated genes for primary tumors wereRAR-β2, RASSF1A, and MGMT, respectively. Surprisingly, RAR-β2 washypermethylated in 70% of the primary tumors. Fifteen (75%) tumors hadone or more genes methylated, and only four (20%) had two or more genesmethylated. Of the metastatic tumors 32 (97%) had one or more genesmethylated, and 17 (52%) had two or more genes methylated (Table 9).Overall, metastatic tumors had a higher frequency of hypermethylatedgenes. Paraffin-embedded histopathology tumor-negative lymph nodes(n=12) were negative for hypermethylation of RAR-β2, RASSF1A, and MGMT(FIG. 1). Analysis of the combination of metastatic (frozen andparaffin-embedded) melanoma tumors (n=86) is shown in Table 9. There wasno significant association of hypermethylation between any individualgenes, nor was gene hypermethylation correlated with disease outcomes.However, RAR-β2 significantly (p=0.009) correlated with primary tumorBreslow thickness. This correlation is important in that Breslowthickness is a major prognostic factor in early stage melanoma patientswith localized disease.

Gene reexpression by demethylation. Expression of MGMT, RASSF1A, andRAR-β2 was assessed in eight melanoma cell lines by RT-PCR. No genetranscripts were detected in cell lines exhibiting hypermethylation.Gene expression was detected in the non-methylated cell lines and insome partially hypermethylated cell lines.

Melanoma cell lines that were hypermethylated were treated with the DNAmethylation inhibitor 5Aza-dC to reverse epigenetic transcriptionalsilencing caused by methylation (16). Melanoma cell lines were treatedwith 0, 3, and 5 μM of 5Aza-dC for four days. Treatment with 5Aza-dCinduced significant elevation of gene expression in all methylated celllines, of which mRNA expression for was absent or very minimal prior todrug treatment (FIG. 2). Melanoma cell lines exhibiting nohypermethylation of the gene promoter region for MGMT and RASSF1Ademonstrated significant elevation of mRNA levels. For assessment ofhypermethylated RAR-β2 in cell lines, cells were treated with 5Aza-dCfor four days followed by treatment with 1 μM ATRA for 24 h. Methylationwas reversed in all treated cell lines and RAR-β2 mRNA expressionelevation was detected. Cell lines demonstrating hypermethylated productby MSP showed no mRNA expression. For some of the genes in the celllines, there was moderate to low levels of hypermethylated productproduced by MSP with minimal gene expression before drug treatment. Geneexpression was significantly elevated in all cell lines after drugtreatment. These in in vitro studies are suggestive thathypermethylation of the promoter region of the genes assessed is anactive mechanism of silencing gene expression.

Bisulfite sequencing was carried out on multiple cell lines to confirmthe methylation status of MGMT, RASSF1A, and RAR-β2 CpG island promoterregions and confirm the MSP results. Representative examples ofsequencing is shown in FIG. 3. Sequencing and MSP of individual genehypermethylation were concordant.

Analysis of circulating methylated DNA in plasma. We examined thepresence of methylated DNA circulating in plasma of melanoma patientsusing realtime PCR. A realtime MSP assay was developed for detection ofmethylated DNA in plasma because the level of DNA is significantly lowerin plasma than tissues. An assay using a reference gene (MYOD1) andstandard curves for individual markers was developed to assess realtimeMSP results. The average DNA recovered from normal donor controls was18.15 ng DNA/500 μl (range 5.31-42.92) and from melanoma patients 29.24ng DNA/500 μl (range 9.97-166.87). In this study, we assessed RAR-β32,RASSF1A, and MGMT, the three genes most frequently hypermethylated intumors. Thirty-one AJCC stage III/IV patients in whom we had pairedplasma and melanoma tumor tissues comprised the study group. Selectionwas based on availability of paired samples. Plasma was obtained from apreoperative blood specimen. The most frequently methylated gene inplasma was RASSF1A (n=6; 19%) followed by MGMT (n=6; 19%) and RAR-β2(n=4; 13%). Of the 31 patients, 29% had at least one genehypermethylated and 16% of the patients had a least two geneshypermethylated in their plasma. Plasma from 33 healthy normal donorswas negative for hypermethylation of all three genes. Analysis ofmethylation for MYOD1 gene was run on all samples for verification ofmodification and detection of DNA. Concordance of plasma genehypermethylation status to respective paired tumors was as follows: MGMT(6 of 17; 33%); RASSF1A (5 of 20; 24%); and RAR-β2 (4 of 20; 18%). Thissuggests that there may be degradation or limited release of these DNAmarkers. In two patients, hypermethylation of RASSF1A was absent intumors but present in plasma. This may be due to other metastases notsurgically excised or subclinical disease. These preliminary studiessuggest hypermethylated genes can be detected in accellular plasma ofmelanoma patients. Further detailed studies will validate the clinicalutility of these DNA markers.

Discussion

Hypermethylation of gene promoter regions silences genes in many typesof carcinomas. Profiling studies have shown gene hypermethylationfrequency and specific genes for tumors of different histologicalorigins (Chen et al 2003; Esteller et al 2001; Maruyanma et al 2001;Pfeifer et al 2002; Toyooka et al 2003; and Widschwendter et al 2002).Patterns of gene hypermethylation in primary tumors versus metastases,and their association with clinicopathological factors are not welldescribed. However, there is clear indication that hypermethylation ofTSG promoter regions is a significant mechanism by which genetranscription is turned off in cancer cells. Studies of tumor cell linesmay not be accurate as to the actual frequency of hypermethylation ofgene promoter regions in tumor specimens (Paz et al 2003; Smiraglia etal 2001; and Ueki et al 2000). Hypermethylation of specific genes incell lines may represent clonal selection during culture adaptation andpassaging. Our major finding is that hypermethylation of promoterregions of known and candidate TSGs in melanomas is quite frequent.Hypermethylation of a gene can be potentially used as a surrogate ofaltered gene expression patterns to characterize phenotypic behavior.

At least one of seven genes was hypermethylated in 93% of cell lines, afrequency that suggest a relation between hypermethylation and melanomaprogression. The study demonstrated that several frequently methylatedTSGs in carcinomas were also found in primary and metastatic cutaneousmelanomas. Interestingly, the three genes most commonly hypermethylatedin cell lines, two were slightly more hypermethylated and one was morehypomethylated in metastatic tumors. Recent studies have reviewed thecomparison of cell lines and tumors and have come up with differentconclusions (Paz et al 2003; Smiraglia et al 2001; and Ueki et al 2000).However, one has to be careful in comparing cell lines to tumors; animportant consideration is whether the tumor is a primary or metastaticlesion or one of multiple lesions. In our study, hypermethylation wasless marked in primary tumors compared to cell lines and metastases. Oneexception was RAR-β2 where both primary and metastatic tumorsdemonstrated higher levels of hypermethylation than cell lines.

RAR-β2 was the most frequent hypermethylated gene in the panel of sevengenes assessment. This is the first major report in describinghypermethylation of RAR-β2 in melanoma in a large series of tumorspecimens. Previous studies have demonstrated the frequenthypermethylation of this gene in breast and lung carcinomas (Paz et al2003; Sirchia et al 2002; and Widschwendter et al 2000). RAR-β2 is amember of the nuclear retinoid receptor of genes, family referred asretinoic acid receptors (RAR) (Mangelsdorf et al 1995), which arefrequently turned off or not expressed in a number of carcinomas. Theloss of RAR-β2 has been implicated in tumorigenesis. Interestingly, thefrequency (70%) of RAR-β2 hypermethylation was similar among primary andmetastatic tumors. This is one of the highest frequencies of geneticaberration reported for sporadic primary melanomas. BRAF mutation inprimary tumors is about 31% (Shinozaki et al; unpublished data). Theinhibition of transcription of RAR-β2 may be a key factor in sporadiccutaneous melanoma tumor development. RAR-β3 loss has been demonstratedas a biomarker of bronchial preneoplasia (Kurie et al 2003). Wedemonstrated a significant correlation between hypermethylation ofRAR-β2 and increasing primary tumor Breslow thickness which is a majorprognostic factor for early-stage melanoma (Bostick et al 1999).Silencing of RAR-β2 may be a key epigenetic factor in melanocytetransformation and primary lesion progression. Further studies areneeded to identify RAR-β2 loss during melanocyte and nevustransformation to melanoma.

Retinoic acid treatment can induce differentiation and inhibition ofproliferation in selective melanoma cells (Demary et al 2001). Thevariable responsiveness of melanomas has not been understood. It hasalso been shown that retinoic acid can activate RAR-β3 receptors(Spanjaard et al 1997). Further studies may be warranted to examinestrategic molecular targeting of therapeutics based on RAR-β3 status inmelanoma.

The second most frequently hypermethylated gene was RASSF1A. This largerstudy supported our previous report that RASSF1A is frequentlymethylated in metastatic melanomas (Spugnardi et al 2003). The 42%higher rate of RASSF1A hypermethylation in metastatic versus primarytumors suggests that hypermethylation of RASSF1A is likely to beacquired during tumor progression. Few published studies have comparedhypermethylation of genes in primary and metastatic tumors of the sametumor type. The functional role of RASSF1A is still not clear. However,its inactivation as a TSG in multiple types of cancers has beendemonstrated (Damann et al 2000; Dammann et al 2001; Lo et al 2001;Pfiefer et al 2002; and Spugnardi et al 2003).

The third most commonly gene hypermethylated gene was MGMT; its rate ofhypermethylation was 24% higher in metastases than in primary tumors.MGMT, a DNA repair gene, serves as a key regulator of genome integrity.Studies have shown that MGMT expression protects mammalian cell linesfrom spontaneous G:C to A:T transitions (Christmann et al 2001).Melanoma is known to have acquired resistance to antineoplastic agentssuch as alklylating drugs exhibiting methylating and chlorethylatingproperties such as dacarbazine, procarbazine, and temozolomide(Christmann et al 2001). The overall frequency of DAPK, p16^(ink4a) andGSTP1 gene often found hypermethylated in carcinomas, was quite low inmelanoma. The three major genes we assessed were frequentlyhypermethylated in both primary and metastatic melanomas. There arelikely other TSGs and tumor-related genes inactivated throughhypermethylation during melanoma progression. A more global screeningapproach is needed such as DNA methylation microarray analysis (Shi etal 2003) that will allow assessment of multiple genes for multiplespecimens.

Previously, we have demonstrated circulating DNA in plasma in the formof LOH of microsatellites in melanoma patients (Fujiwara et al 1999 andTaback et al 2001). In the present study, we demonstrated that melanomapatients have circulating hypermethylated DNA in their plasma. The threemost common genes in melanoma tumors were detected in plasma at a lowerfrequency. A quantitative realtime PCR assay was developed to improvesensitivity and accuracy of methylated DNA in plasma. The assay was 100%specific as no normal donors' plasma under the assay conditions waspositive. Future studies need to optimize the assay to obtain highsensitivity for early disease diagnosis. This is the first major studydemonstrating the presence of a significant number of melanoma patientswith circulating methylated DNA markers. The half-life of individualgenes will play a significant role as to the value of detection of thesecirculating DNA. Circulating methylation DNA markers may be used assurrogates of subclinical disease recurrence or progression. Detailedstudies on larger cohorts of patients are needed to determine whetherthese circulating methylation markers have clinical utility inpredicting disease outcome. Nevertheless, it is intriguing thatcirculating methylated DNA is present in plasma and released by tumorcells. Whether this DNA is from established metastases or circulatingtumor cells in blood needs to be determined. Further studies on definedcohorts of melanoma patients need to be studied to determine thepotential clinicopathological utility in assessment of melanoma patientsplasma for circulating DNA.

Example 4 Prognostic Significance of Hypermethylated Tumor SuppressorGenes in Metastatic Melanomas Introduction

Hypermethylation (HM) of CpG islands of promoter regions of tumorsuppressor genes (TSG) silences gene expression and promotes tumorprogression. Among AJCC stage III melanoma patients who have palpablenodes and undergo complete lymph node dissection (CLND), there arepatients who have better prognosis than others even when matched forprognostic factors. We have discovered that the TSG RAS associationdomain family protein 1 (RASSF1A) and retinoic acid receptor (32 (RARB)are HM in cutaneous melanomas. We hypothesized that regional lymph nodemetastasis with HM TSGs is predictive of a poorer disease outcome.

Methods

AJCC stage III melanoma patients (n=37) who underwent CLND with palpablenodes were selected by the biostatistician. HM of the promoter regionsof RASSF1A and RARB using quantitative realtime methylation-specific PCRfrom DNA isolated of paraffin-embedded metastatic tumors was analyzed.Genomic copy numbers of gene methylation were normalized and quantifiedwith copy numbers of the MYOD gene.

Results

The study group consisted of 10 females and 27 males (mean age 55.7 yrs;mean Breslow thickness 2.32 mm±1.46). Primary lesions were located onthe trunk (14), extremities (15), head and neck (5), or an unknown site(3). All patients had at least 2 palpable nodes (mean 11.4; range 3-33).HM was detected for RASSF1A alone (16%), RARB alone (28%), and both TSGs(14%). HM RARB alone correlated with overall survival and disease-freesurvival by multivariate analysis, Wald test; p=0.008 and p=0.009,respectively. The median overall survival was 27.7 mos for nonHM RARB vs9.5 mos for HM RARB. The median disease-free survival was 8.5 mos fornonHM RARB vs 3.9 mos for HM RARB. RARB did not correlate with any ofthe 7 prognostic factors.

Conclusions

HM of RARB in regional lymph node metastasis has prognostic significancein prediction of disease outcome in melanoma patients. This pilot studydemonstrates that epigenetic inactivation of TSG can be used as genomicpredictive marker of disease outcome.

Example 5 Blood, Bone Marrow, and Tumor Markers for Various Types ofCancer Materials and Methods

BM Sample Preparation. Bone marrow was drawn and (cell-free supernatant)plasma was immediately separated by centrifugation (1000×g, 15 min),filtered through a 13-mm serum filter (Fisher Scientific, Pittsburgh,Pa.) to remove any potential contaminating cells, aliquoted andcryopreserved at −30° C. For normal genomic DNA controls, whole bloodwas collected from each patient spotted and stored on FTA blood cards(Fitzco, Minneapolis, Minn.) prior to DNA isolation. DNA was extractedfrom one ml of BM aspirate plasma using QIAamp extraction kit (Qiagen,Valencia, Calif.) using conventional methods (Taback et al 2001).

Preparation of Samples from Primary Tumor Tissues and LOH Analysis ofthe Obtained Samples. DNA was isolated from 10 μm sections cut fromparaffin-embedded tumor tissue blocks. Samples were deparaffinized andmicrodissected using laser capture microscopy (Arcturus, Mountain View,Calif.) from normal tissue. Microdissection may also be carried outusing a scalpel or needle and a microscope or a precision laser cuttinginstrument. Then, DNA was isolated, processed, purified, andquantitated.

For example, in one study, DNA was isolated by incubating the sampleswith proteinase K in lysis buffer (50 mM Tris-HCl, 1 mM EDTA and 0.5%Tween 20) at 37° C. overnight and then heated at 95° C. for 10 min. Theobtained samples were amplified and analyzed for LOH using PCR methods.The amplification/detection methods used were PCR and gelelectrophoresis using labeled primers (fluorescent or radioactive);RealTime PCR using specific labeled primers Taqman and probes (labeledwith chromatographic dyes); or capillary array electrophoresis (CAE)with labeled PCR primers (no probes). All of these methods are known tothose skilled in the art and will not be described here in detail.

LOH Analysis in Blood and BM Samples. LOH analysis of blood(plasma/serum) and bone marrow were performed as described in papers byB. Taback (Cancer Res 2001) and Y. Fujiwara (Cancer Res 1999), thecontent of which is incorporated herein by the reference. DNA wasisolated, processed, purified, and analyzed for the presence of LOH asgenerally described in B. Taback and Y. Fujiwara references. Theisolation procedure was the same regardless the type of cancer (breast,melanoma, prostate, colon cancer) being detected.

The amplification/detection methods used were PCR and gelelectrophoresis using labeled primers (fluorescent or radioactive);RealTime PCR using specific labeled primers Taqman and probes (labeledwith chromatographic dyes); or capillary array electrophoresis (CAE)with labeled PCR primers (no probes). All of these methods are known tothose skilled in the art and will not be described here in detail.

Methylation Analysis of Blood, BM, and Tumor Tissue Samples. The samplesof blood, BM, and tumor tissue were prepared as the samples for LOHanalysis. Then, the samples were treated with bisulphite and proteinaseK to separate out methylated from unmethylated DNA. Methylation specificPCR (MSP) was performed. A more detailed description of this methodfollows.

DNA was isolated from cell lines and tissues using DNAzol Genomic DNAIsolation Reagent (Molecular Research Center, Inc., Cincinnati, Ohio)according to the manufacturer's recommendations. The methylation statusof the marker promoter region was determined by a bisulfite modificationprotocol^(60,61). Briefly, 1 mg of genomic DNA was denatured in NaOH at37° C. Cytosines were sulfonated in the presence of sodium bisulfite and5 mM hydroquinone (Sigma) in a water bath for 16-18 h at 55° C. The DNAsamples were desalted using the Wizard DNA Clean-Up System (Promega,Madison, Wis.) and desulfonated in NaOH at 37° C. Treated DNA sampleswere precipitated with ethanol and resuspended in 10 mM Tris-Cl, 1 mMEDTA, pH 7.6. DNA sequences were amplified by mixing 100 ng of bisulfitetreated DNA with 50 pmoles of individual primer sets; reaction buffercontaining each dNTP and Taq polymerase at 95° C. for 1 min, 55° C. for1 min and 74° C. for 2 min for 30 cycles.

For MSP (methylation specific PCR), two methods were used to assess thedifferent regions of the marker CpG promoter island. In the firstmethod, PCR was performed and assessed on on 2% Tris-borate EDTA agarosegel. In the second method, one hundred ng of bisulfite-modified DNA wasamplified in a final reaction volume of 20 ul containing 0.8 mM dNTPs,and Taq polymerase. PCR was performed with an initial 10 min incubationat 95° C., followed by 40 cycles of denaturation at 95° C. for 30 sec,annealing at 60° C. for 30 sec, and extension at 72° C. for 30 sec, anda final 7 min hold at 72° C. PCR products were visualized usingcapillary array electrophoresis (CAE; CEQ 2000XL DNA Analysis System,Beckman Coulter, Fullerton, Calif.).

The assay was set up in a 96-well microplate format. Multiple PCRproducts can be run in each well for comparisons. Multiple PCR productswere visualized simultaneously by labeling forward primers with a choiceof three Beckman Coulter WellRED Phosphoramidite (PA)-linked dyes.Forward methylated specific primer was labeled with D4pa dye (blue) andforward unmethylated specific primer was labeled with D2pa dye (black).One ml of methylated PCR product and one ml of unmethylated PCR productwere mixed with 40 ml loading buffer and 0.5 ul dye-labeled sizestandard (Beckman Coulter Inc.). Labeling forward primers specific formethylated or unmethylated modified DNA distinguishes the respectiveproducts so that they may be analyzed simultaneously. Other methods fordetection of methylation markers, such as ligase PCR and Realtime PCRwith specific marker probe, may also be used.

Methylation Site Sequencing to Prove the Marker's Site is Methylated.Bisulfite sequencing was carried out to confirm the methylation statusof the CpG island promoter region, which regulates the marker genetranscription. Extracted DNA was treated with sodium bisulfite, whichconverts unmethylated cytosines to uracil. Thymine is then substitutedfor uracil during subsequent PCR. Methylated cytosines(5-methylcytosine) are protected from this process and remain unchanged.Accordingly, all cytosines present following sequence analysis representmethylated cytosines. All markers used for methylation were confirmed bymethylation sequencing.

Results

Determining LOH in the BM of Breast Cancer Patients. BM aspirates werecollected in 4.5 ml sodium citrate tubes (Becton Dickinson, FranklinLakes, N.J.) through bilateral anterior iliac approach from 48consecutive patients as follows: ductal carcinoma in situ (DCIS), 1patient; American Joint Committee on Cancer (AJCC) stage I, 32 patients;AJCC stage II, 13 patients; and AJCC stage III, 2 patients; undergoingsurgical resection of their primary breast cancer at the Saint John'sHealth Center/John Wayne Cancer Institute. In addition, five healthyfemale volunteer donors contributed BM aspirate samples for controls. Toassess the correlation of LOH found in the BM and that of the primarybreast tumor, DNA was isolated from 10 μm sections cut fromparaffin-embedded tumor tissue blocks. Additionally, each BM aspiratewas assessed for the presence of occult tumor cells by conventionalhistologic staining methods using Hematoxylin and Eosin (H&E).

Eight polymorphic microsatellite markers which correspond to regionsthat have been shown to demonstrate significant LOH suggesting sites ofputative tumor suppressor and/or metastasis related genes were selected:D1S228 at 1p36; D8S321 at 8qter-8q24.13; D10S197 at 10p12; D14S51 at14q32.1-14q32.2; D14S62 at 14q32; D16S421 at 16q22.1; D17S849 at17pter-17qter, and D17S855 at 17q. All primer sets were obtained fromResearch Genetics (Huntsville, Ala.) and sense primers were labeled witha fluorescent dye: 5-(and-6)-carboxyfluoroscein, FAM.

Approximately 20 ng of genomic DNA was amplified by PCR in 25 μlreactions containing 1×PCR buffer (Perkin Elmer, Foster City, Calif.), 6μmol of each primer, 1 unit of Taq DNA polymerase, 2.5 μMdeoxynucleotide triphosphates, and 1.5 mM MgCl₂. Forty PCR cycles wereperformed with each cycle consisting of 30 at 94° C., 30 s at 50-56° C.,and 90 s at 72° C., followed by a final extension step of 72° C. for 5min as previously described (Taback et al 2001).

PCR products were electrophoresed on 6% denaturing polyacrylamide gelcontaining 7.7 M urea at 1600V for 2 h. Genomyx SC scanner (BeckmanCoulter, Fullerton, Calif.) was used to image the fluorescent-labeledPCR products and densitometric analysis was performed with ClaritySCsoftware (Media Cybernetics, Silver Spring, Md.). Intensity calculationsand comparisons of the specific alleles in patients' normal control andrespective BM DNA were performed to evaluate for LOH. The LOH wasdefined if a greater than 50% reduction of intensity was noted in oneallele from tumor or BM DNA when compared with the respective allele inthe matched-paired lymphocytes (Taback et al 2001).

Clinical and histopathologic data was obtained from patient chart reviewand the Breast Tumor Registry at the John Wayne Cancer Institute.Chi-Square and Wilcoxon Rank Sum tests were performed for statisticalevaluation of association of BM LOH status and known prognosticparameters.

LOH was identified in 11 (23%) of 48 patients' BM aspirates. LOH wasmost commonly identified at microsatellite marker D14S62 occurring in 4(12%) of 34 informative patients. Microsatellite markers demonstratingLOH at D1S228 and D14S51 occurred in 3 (8%) of 38 informative patientseach, followed by LOH at D8S321 (5%), D10S197 (4%), and D17S855 (3%). NoLOH was detected for microsatellite markers D16S421 and D17S849 (Table10). Eight of the 11 patients with detectable LOH in their BMdemonstrated this event at only one of the chromosome loci assessed andthree patients (1 stage I, 2 stage II patients) contained LOH for twomicrosatellite markers. No LOH was detected for any of themicrosatellite markers assessed in the patient with DCIS or the BMaspirates collected from five healthy female donors.

TABLE 10 LOH Frequency in Breast Cancer Patients Bone Marrow AspiratesMicrosatellite marker LOH in BM aspirates/informative cases (%) D14S62 4/34 (12%) D14S51 3/38 (8%) D1S228 3/38 (8%) D8S321 2/39 (5%) D10S1971/26 (4%) D17S855 1/37 (3%) D17S849 0/31 (0%) D16S421 0/28 (0%)

The inventors found LOH on chromosome 14q as the most frequent eventidentified on circulating DNA in BM. However, in another study, LOH on14q has been shown to occur more commonly in primary tumors withoutlymph node metastasis, suggesting a site for a possiblemetastasis-related gene; however, the metastasis itself was not assessedfor LOH(O'Connell et al 1999). While not wanting to be bound by atheory, the inventors believe that metastatic clones at different sitesmay demonstrate different LOH profiles. Additionally, differences inthese results may reflect the stability of this marker as detected fromvarious sources (blood, BM, tumor tissues) or it may be uniquelyassociated with site-specific metastasis. Molecular markers that arespecific for the metastatic phenotype and/or sites of metastasis mayprove useful for focusing clinical assessments.

There was an increased association between the presence of LOH in the BMand advanced disease stage. Six (19%) of 32 AJCC stage I patientsdemonstrated LOH for at least one marker, in contrast to 4 (31%) of 13AJCC stage II patients, and 1 (50%) of 2 AJCC stage III patients (Table11).

TABLE 11 Association of LOH in Patients' BM Aspirates with AJCC StageAJCC Stage Patients with LOH in BM/total patients (%) I 6/32 (19%) II4/13 (31%) III  1/2 (50%)

Ten clinicopathologic prognostic factors were assessed for correlationwith BM LOH status: histologic tumor type, size, grade, Bloom-Richardsonscore, lymph node involvement, presence of lymphovascular invasion inthe primary tumor, receptor status (estrogen, progesterone, HER2), andp53 status. There was an association between larger tumor size and BMLOH positivity: 2.46 cm versus 1.81 cm, mean tumor sizes, respectively.There was also a trend towards an increased incidence of BM LOH in lymphnode positive patients as compared with lymph node negative patients: 3(33%) of 9 patients versus 8 (21%) of 38 patients, respectively. Nosignificant correlation existed between any prognostic factor and BM LOHstatus in this study except histology. Lobular carcinomas were morelikely associated with increased LOH in BM aspirates than infiltratingductal tumors: 6 (60%) of 10 patients versus 5 (14%) of 37 patients,respectively (Chi-Square test P=0.006). Larger populations withlong-term follow-up are warranted to evaluate the clinical andprognostic utility of this assay.

To determine whether a correlation existed between the LOH detected inpatients BM and their primary tumor, DNA was isolated from primarytumors and evaluated with identical microsatellite markers. Ten of theeleven patients demonstrating LOH in their BM had primary tumorsavailable for assessment. In all ten patients, the LOH identified in theBM was also present respectively in the primary tumor (FIG. 4).Conventional histological analysis of all specimens using standard H&Estaining did not demonstrate occult tumor cells in any of the BMsamples.

Determining LOH and CpG Hypermethylation in Blood (Serum/Plasma) ofProstate Cancer Patients. To evaluate the clinical utility of assessingcirculating nucleic acids containing tumor-associated geneticalterations in serum/plasma of prostate cancer, patients' blood wascollected from 23 prostate cancer patients American Joint Committee onCancer (AJCC) stages I-IV. A panel of 7 microsatellite markers (D8S261,D8S262, D9S171, D10S591, D10S532, D16S422 and D18S70) on 6 chromosomearms pertaining to putative tumor suppressor gene regions was utilizedto assess prostate cancer patient's serum/plasma for LOH on circulatingnucleic acids.

In addition, methylation specific PCR and capillary arrayelectrophoresis was performed on the same samples to evaluate thepresence of CpG island hypermethylation in the promoter regions of threeknown tumor suppressor genes: RASSF1A, RAR-β3 and GSTP1. The obtainedresults are shown in Table 12 below.

TABLE 12 Association of LOH and Methylation in Prostate Cancer Patients'Blood Samples with AJCC Stage AJCC Stage Methylation LOH MolecularPositive (%) I 0/3 0/3 0% II 0/5 2/5 40% III/IV  8/15  5/15 60%

A correlation of an increased combination of promoter regionhypermethylation and LOH identified on circulating nucleic acids wasassociated with advancing AJCC staging. The incidence of LOH increasedfrom 2 (25%) of 8 stage I-II patients to 5 (33%) of 15 stage III-IVpatients. None of the early stage patients demonstrated methylated DNAin their blood whereas 8 (53%) of 15 stage III-IV patients wherepositive for any one methylated marker in their blood. Although, itappears that testing blood for hypermethylation or LOH alone may be usedto detect and diagnoses prostate cancer, testing for bothhypermethylation and LOH may provide a higher sensitivity and accuracyin detection and determination of the AJCC stage of prostate cancer.

Determining LOH and CpG Hypermethylation in Blood (Serum/Plasma) ofColorectal Cancer Patients. To evaluate the presence of circulatingnucleic acids containing tumor-associated genetic alterations inserum/plasma of colorectal cancer patients, blood was collected from 33colorectal cancer patients undergoing surgical resection for theirprimary diagnosis. A panel of 11 microsatellite markers (D4S175,D4S1586, D5S299, D8S133, D8S264, D15S127, TP53, D17S796, D17S1832,D18S58 and D18S61) on 6 chromosomes at loci demonstrating frequent LOHin primary colorectal tumors suggestive of putative tumor suppressorgene regions were utilized. In addition, methylation specific PCR andcapillary array electrophoresis was performed on the same samples in 16patients to evaluate the presence of CpG island hypermethylation in thepromoter regions of five known tumor suppressor genes: MGMT, P16, APC,RASSF1A and RAR-β3. The obtained results are shown in Tables 13 and 14below.

TABLE 13 Frequency of Microsatellite Marker LOH in Colorectal CancerPatients' Blood D18S58 1/23 (4%) D18S61 1/32 (3%) TP53 2/28 (7%)D17S1832 2/28 (7%) D5S299  5/25 (20%) D4S175  6/23 (26%) D4S1586 1/19(5%) D8S133  3/19 (16%) D8S264 1/22 (5%) D15S127 2/29 (7%) D17S796  3/22(14%)

TABLE 14 Frequency of Gene Promoter Methylation in Colorectal CancerPatients' Blood Methylation MGMT P16 RAR-β RASSF1A APC 2/16 (13%) 0/16(0%) 0/16 (0%) 1/16 (6%) 4/16 (25%)

In this study LOH, for any one marker, was identified in 17 (52%) of 33patients blood samples (Table 13) and promoter region hypermethylationat any marker was detected in 6 (38%) of 16 patients (Table 14). Thecombination of hypermethylation and LOH for assessing blood in those 16patients evaluated using both techniques identified a greater number ofpatients with colorectal cancer, 15 (94%) of 16 as compared to eithermethod alone: methylation 6 (38%) or LOH 11 (69%) of 16 patients (Table15).

TABLE 15 Frequency of Methylation, LOH, and Their Combination inColorectal Cancer Patients' Blood Assessed Using Both Assays MethylationTotal + − LOH LOH + 2 9 11 − 4 1 5 Total 6 10 Methylation

Detection of LOH and Methylation Markers in Primary Breast Cancer Tumorsto Predict Metastasis to Lymph Nodes and Disease Outcome. MethylationPCR was carried out on primary tumors (151 patients) for the differentmarkers. Patients were divided into those that had no metastasis intheir sentinel lymph node (first tumor draining lymph node), occult ormicrometastasis in their lymph nodes, or had palpable (clinicallydetectable) metastasis in their lymph nodes. Preliminary analysisdemonstrated that methylation of specific markers in the primary tumorcan predict the type of metastasis in the nodes. The results shown inTable 16 indicate that additional diagnostic data may be obtained fromlymph node metastasis samples.

TABLE 16 Methylation Status of Primary Tumor and Lymph Node (LN)Metastasis (29 Pairs) E- Primary-LN met APC GSTP1 RASSF1A RAR-b cadTWIST* M-M 8 7 16 9 14 7 M-U 7 3 7 5 1 5 U-M 2 0 1 5 12 1 U-U 12 19 5 102 15 M stands for methylated and U stands for unmethylated

Inventors believe that the same markers can be used to analyze blood(plasma/serum) and BM samples of melanoma patients.

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While the foregoing has been described in considerable detail and interms of preferred embodiments, these are not to be construed aslimitations on the disclosure or claims to follow. Modifications andchanges that are within the purview of those skilled in the art areintended to fall within the scope of the following claims. Allliteratures cited herein are incorporated by reference in theirentirety.

1. A method of detecting DNA markers in a sample, comprising: providinga blood sample from a subject; and detecting one or more DNA markers inthe sample, wherein the DNA markers are indicative of LOH or DNAhypermethylation, or the DNA markers are indicative of DNA mutation inKRAS or BRAF gene.
 2. The method of claim 1, wherein the DNA markers arein the 1p, 3p, 6p, 6q, 8p, 10q, 11q, 14q, 16q, or 17p region.
 3. Themethod of claim 1, wherein the DNA markers include D1S228, D8S321,D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,D10S197, D10S591, D10S532, D14S51, D14S62, D15S127, D16S421, D16S422,D17S796, D17S849, D17S855, D18S58, D18S61, or D18S70.
 4. The method ofclaim 1, wherein the DNA markers are indicative of hypermethylation inRASSF1A, MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK, P16, or Cyclin D2promoter.
 5. A method of detecting cancer, comprising providing a bloodsample from a subject; and detecting the presence or absence of LOH, DNAhypermethylation, mutation or a combination thereof of one or more DNAmarkers in the sample; and determining that the presence of LOH, DNAhypermethylation, mutation or a combination thereof of the one or moreDNA markers is indicative of cancer in the subject.
 6. The method ofclaim 5, wherein the markers include KRAS or BRAF, and mutation of themarkers is indicative of cancer in the subject.
 7. The method of claim6, wherein the cancer is melanoma, neuroblastoma, colorectal, breast, orprostate cancer.
 8. The method of claim 5, wherein a combination of LOHand DNA hypermethylation of the markers is indicative of melanoma,neuroblastoma, colorectal or prostate cancer in the subject.
 9. Themethod of claim 8, wherein the presence of absence of LOH is indicatedby one or more DNA markers that include D1S228, D8S321, D4S175, D4S1586,D5S299, D8S133, D8S261, D8S262, D8S264, D9S171, D10S591, D10S532,D14S51, D14S62, D15S127, D16S421, D16S422, D17S796, D17S849, D17S855,D18S58, D18S61, or D18S70.
 10. The method of claim 8, wherein thepresence or absence of DNA hypermethylation is detected in RASSF1A,MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK, or Cyclin D2.
 11. A method ofstaging cancer, comprising providing a blood sample from a subjectsuffering from cancer; and detecting the presence or absence of LOH, DNAhypermethylation, mutation or a combination thereof of one or more DNAmarkers in the sample; and determining that the presence of LOH, DNAhypermethylation, mutation or a combination thereof of the one or moreDNA markers is indicative of an advanced stage of the cancer in thesubject.
 12. The method of claim 11, wherein the cancer is melanoma,neuroblastoma, colorectal, breast, or prostate cancer.
 13. The method ofclaim 11, wherein a combination of LOH and DNA hypermethylation of themarkers is indicative of an advanced stage melanoma, neuroblastoma,colorectal or prostate cancer in the subject.
 14. The method of claim13, wherein the presence or absence of LOH is indicated by one or moreDNA markers that include D1S228, D8S321, D4S175, D4S1586, D5S299,D8S133, D8S261, D8S262, D8S264, D9S171, D10S591, D10S532, D14S51,D14S62, D15S127, D16S421, D16S422, D17S796, D17S849, D17S855, D18S58,D18S61, or D18S70.
 15. The method of claim 13, wherein the presence orabsence of DNA hypermethylation is detected in RASSF1A, MGMT, GSTP1,RAR-.beta., TWIST, APC, DAPK, or Cyclin D2.
 16. A method of prognosingcancer, comprising providing a blood sample from a subject sufferingfrom cancer; and detecting the presence or absence of LOH, DNAhypermethylation, mutation or a combination thereof of one or more DNAmarkers in the sample; and determining that the presence of LOH, DNAhypermethylation, mutation or a combination thereof of the one or moreDNA markers is indicative of a poor prognosis of the cancer in thesubject.
 17. The method of claim 16, wherein the cancer is melanoma,neuroblastoma, colorectal, breast, or prostate cancer.
 18. The method ofclaim 16, wherein a combination of LOH and DNA hypermethylation of themarkers is indicative of a poor prognosis of the cancer in the subject.19. The method of claim 18, wherein the presence or absence of LOH isindicated by one or more DNA markers that include D1S228, D8S321,D4S175, D4S1586, D5S299, D8S133, D8S261, D8S262, D8S264, D9S171,D10S591, D10S532, D14S51, D14S62, D15S127, D16S421, D16S422, D17S796,D17S849, D17S855, D18S58, D18S61, or D18S70.
 20. The method of claim 18,wherein the presence or absence of DNA hypermethylation is detected inRASSF1A, MGMT, GSTP1, RAR-.beta., TWIST, APC, DAPK, or Cyclin D2.